Compositions, solutions, and methods used for transplantation

ABSTRACT

This invention discloses a method for reducing the intracellular lipid storage material of a cell, tissue, or organ for transplantation and features solutions, methods and kits that induce the metabolic elimination of lipid storage in a cell, tissue, or organ. In one exemplary approach, the process involves contacting a cell, tissue, or organ with a perfusate solution that include catabolic hormones and amino acids, at physiological conditions, to increase lipid export and lipid oxidation. If desired, the cell, tissue, or organ of the invention may also be heat shock preconditioned. The invention can be used to prepare, recondition, or store a cell, tissue, or organ for transplantation by increasing tolerance to ischemia-reperfusion and cold-preservation related injury.

BACKGROUND OF THE INVENTION

In general, the present invention relates to cell, tissue, and organtransplantation.

Currently, a major limitation of clinical transplantation is thepersistent shortage of organs, which results in an extensive number ofpatients being placed on wait-lists. Furthermore, a large proportion ofpatients die even before a suitable transplant can be found.

In the context of liver transplantation, although the majority of liverdonors are cadaveric, living and split liver donor techniques arepromising alternatives, yet represent only about 3% of the total numberof transplants performed in the United States (Sindhi et al. J Ped.Surg. 34: 107-110, 1999). Furthermore, living donor methods areinherently limited because they represent a significant risk to thedonor. Another approach is the use of bioartificial liver supportsystems, which may provide temporary liver function support and, incases in which the patient recovers from the acute phase of the disease,may avoid the need for a liver transplant altogether. However, in lightof the early stages of development of such strategies, transplantationsinvolving cadaveric organs are likely to remain the mainstay for thetreatment of organ dysfunction for the foreseeable future.

Further exacerbating the problem of organ shortage is the fact that asignificant proportion of donor livers are steatotic or fatty and as aresult, often deemed unacceptable for transplantation purposes. Asignificant number of donor organs are therefore discarded andeliminated from the donor pool even before transplantation. Althoughusually asymptomatic, the accumulation of lipid in livers, also known ashepatic steatosis, is the most common single predisposing risk factorfor postoperative liver failure and accordingly, approximately 65% oflivers rejected for transplantation are steatotic (Urena et al., WorldJ. Surg. 22: 837-844, 1998). In fact, it is noteworthy that no singleother liver pathology is as prevalent as steatosis and is associatedwith such a negative impact on the current shortage of donor livers.

Indeed, data from animal models suggest that steatotic livers are farmore susceptible to ischemia-reperfusion (I/R) related damage thanso-called lean livers. In this respect, I/R causes necrosis andapoptosis of hepatocytes and endothelial cells through the generation ofoxygen reactive species and the disruption of the microvasculature,ultimately leading to hepatic failure. Studies on the effect of coldstorage of liver followed by rewarming and perfusion also show moreextensive damage in fatty livers and a reduced “safe” preservation timebefore transplantation. In the context of liver transplantation, lipidaccumulation in the liver also impairs certain key liver functionsnamely glucose production and cytochrome p450 detoxification activity(Gupta et al., Am. J. Physiol. 278:E985-E991, 2000; Leclercq et al.,Biochem. Biophys. Res. Commun. 268: 337-344, 2000).

Furthermore, livers with mild to moderate steatosis, which areconsidered marginally acceptable, have a lower graft survival rate (76%vs. 89% for lean livers) at four months post-transplantation. Inaddition, patients receiving steatotic livers have a mere 77% survivalrate at two years post-transplantation in comparison to a 91% survivalin patients receiving nonsteatotic livers.

It is therefore clear that methods that salvage or recondition donorlivers discarded because of severe steatosis or that increase thesuccess rate of transplanted steatotic livers would significantly reducethe number of patient deaths and help bridge the gap that exists betweensupply and demand in liver transplantation.

SUMMARY OF THE INVENTION

As is described in greater detail herein, the present invention providesmethods and compositions to prepare a donor cell, tissue, or organ fortransplantation into a recipient involving the metabolic reduction ofintracellular lipid storage in the tissue or organ. It is useful becauseit provides for an efficient means to rapidly remove excess lipidstorage from virtually any potential source of donor material (such as acell, tissue, or organ) which is deemed unacceptable for transplantationdue to its high fat content. In this particular respect, the presentinvention is particularly useful to recondition steatotic organs fortransplantation, for example. If desired, heat shock preconditioning ofthe cell, tissue, or organ may also be used for example, to increase theoverall ability of the cell, tissue, or organ to withstandischemia-reperfusion injury. Overall, the present invention hasimportant applications to transplantation because it significantlyincreases the pool size of available donor material and, as a result,alleviates the current severe shortage of such material, including donorlivers. This, in turn, translates into a reduction in the number ofpatients on the liver transplant waiting list and the number of patientsdying before a suitable transplant is found.

Accordingly, in one aspect, the invention features a method forpreparing a donor cell, tissue, or organ for transplantation into arecipient. This method involves reducing the intracellular lipid storagematerial of the cell, tissue, or organ. In preferred embodiments, ahuman liver cell, human liver tissue, or a human liver organ isprepared.

Preferably, the method of reducing intracellular lipid storage material(e.g., a triglyceride, cholesterol, cholesterol ester, or phospholipid)includes contacting the cell, tissue, or organ with a solution (such asthe defatting solution described herein) that increases oxidation of alipid; increases export of a lipid from the cell, tissue, or organ; orboth. In preferred embodiments, the method results in reducing anischemia-reperfusion injury in the cell, tissue, or organ upontransplantation into a recipient or results in reducing acold-preservation-related injury in the cell, tissue, or organ upontransplantation into a recipient. In other preferred embodiments, themethod reconditions a steatotic cell, tissue, or organ.

If desired, heat shock may also be induced in the cell, tissue or organof the invention. Heat shock may result from increasing the temperatureof the cell, tissue, or organ by at least 1° C. for a period of at leastone minute. For example, the temperature may be increased for a periodranging between one minute and one hour, preferably between 1 minute and30 minutes, and more preferably between 1 minute and 15 minutes.Desirably, the temperature of the cell, tissue, or organ is increased toa range between 37° C. and 50° C., preferably between 38° C. and 45° C.,more preferably between 40° C. and 43° C., and most preferably between42° C. and 43° C.

According to this invention, the increase in temperature may result fromheating the whole body or, alternatively, may result from heating alocalized area of the donor cell, tissue, or organ. The heating may bemediated by placing the cell, tissue, or organ in a solution (e.g., adefatting solution or saline that has been heated to 42° C.) thatinduces heat shock; by perfusing the tissue or organ with a solutionthat induces heat shock; or by warming the blood percolating thelocalized area in which the cell, tissue, or organ is located. Theincrease in temperature may also result from heating the cell, tissue,or organ ex vivo. In general, heating may be mediated by microwave orultrasound treatment.

Alternatively, heat shock may be induced by contacting the cell, tissue,or organ with an agent that increases the expression of at least oneheat shock protein. For example, the cell, tissue, or organ may becontacted with an agent such as cobalt protoporphyrin orgeranylgeranylacetone. Optionally, the cell, tissue, or organ isadministered with a therapeutically effective amount of a heat shockprotein or is provided with at least one expression vector containing anucleic acid sequence encoding a heat shock protein in a therapeuticallyeffective amount. Preferably, the expression of the heat shock proteinis increased by at least 10%, 20%, preferably at least 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 100%, or more than 100% relative to anuntreated control. Exemplary heat shock proteins include HSP72, HSP70,HO-1, and HSP90.

According to this invention, heat shock preconditioning preferablydecreases the proliferation and activation of T cells and decreases theproduction of inflammatory cytokines (e.g., IL-12, Il-10, IFN γ, andTNF-α.). In this regard, CD4⁺ T cells, for example, produce inflammatorycytokines, activate Kuffpner cells, and recruit neutrophils.

If desired, the cell, tissue, or organ may be contacted with acomposition containing gadolinium chloride (GdCl₃) or an agent thatinhibits the T cell proliferation, T cell activation, or both. Such anagent may include, for example, cyclosporine A (CyA) and FK506.

The cell, tissue, or organ that has been preconditioned (defatted, heatshock preconditioned, or both) according to this invention is preferablytransplanted between 3 to 48 hours, between 6 to 48 hours, or 24 hoursafter being prepared. If the donor material is not used fortransplantation, the donor cell, tissue, or organ may be stored,preferably at 4° C.

In another aspect, the invention features a solution (e.g., a defattingsolution) for reducing intracellular lipid storage material of a donorcell, tissue, or organ for transplantation into a recipient; thissolution includes a catabolic hormone and an amino acid. In preferredembodiments, the catabolic hormone of the solution increasesintracellular lipid oxidation; lipid export; or both. Exemplarycatabolic hormones include glucagon, epinephrine, growth hormone,hepatocyte growth factor, leptin, adiponectin, metformin, thyroidhormone, or a glucocorticoid hormone (such as a hydrocortisone, acortisol, a corticosterone, or dexamethasone). In still other preferredembodiments, an amino acid (such as alanine or glutamine) is requiredfor the synthesis of an apolipoprotein. In yet other preferredembodiments, the solution further includes an anti-oxidant or an oxygencarrier. Exemplary anti-oxidants include N-acetyl-cysteine, glutathione,allopurinol, S-adenosyl-L-methionine (a precursor of glutathione),polyphenols (found, for example, in green tea), free iron scavengers(e.g., deferoxamine), adenosine, or inhibitors of inducible nitric oxidesynthase (iNOS) (e.g., N(G)-nitro-L-arginine methyl ester andaminoguanidine) and exemplary oxygen carriers include hemoglobin or aperfluorocarbon. If desired, the solution optionally includes acomponent that provides oncotic pressure.

In preferred embodiments, the solution includes: from 50 mM to 150 mMsodium ion; from 0.4 mM to 4 mM potassium ion; from 0 mM to 50 mMphosphate ion; from 0 mM to 44 mM bicarbonate ion; from 0.19 mM to 5 mMcalcium ion; from 0.081 mM to 5 mM magnesium ion; from 0.2 mM to 2.4 mMalanine; from 0.2 mM to 10 mM glutamine; from 50 pg/mL to 1000 pg/mLglucagon; from 100 pg/mL to 2500 pg/mL epinephrine; from 50 ng/mL to1500 ng/mL hydrocortisone; and from 30 g/mL to 120 g/mL hydroxyethylstarch.

In still other preferred embodiments, the solution includes: 116 mMsodium ion; 2.3 mM potassium ion; 1.0 mM sodium phosphate (monobasic);26 mM sodium bicarbonate; 1.9 mM calcium ion; 0.81 mM magnesium ion;0.48 mM alanine; 2.00 mM glutamine; 100 pg/mL glucagon; 250 pg/mLepinephrine; 150 ng/mL hydrocortisone; and 60.0 g/mL hydroxyethylstarch.

Preferably, the solution is heated to a temperature of 25° C. to 45° C.,preferably 25° C. to 43° C., even more preferably 42° C. to 43° C. or37° C.; is exposed to 20 to 100% O₂, such as 95% O₂; is exposed to 0 to10% CO₂, such as 5% CO₂; and has a pH of 6.5 to 7.8, such as a pH of7.4. Optionally, the solution further contains an agent that increasesthe expression of at least one heat shock protein in a cell, tissue, ororgan, such as cobalt protoporphyrin or geranylgeranylacetone.

In still another aspect, the invention features a method for preparing adonor cell, tissue, or organ (including steatotic cells, tissues, ororgans) for transplantation into a recipient that includes contactingthe donor cell, tissue, or organ with any of the aforementionedsolutions. Preferably, the donor cell, tissue, or organ is contacted forat least 10 minutes, 1 hour, 6 hours, 24 hours, or 48 hours.

Additionally, the invention features a method of storing or preserving adonor cell, tissue, or organ for transplantation into a recipient. Thismethod includes contacting the donor cell, tissue, or organ with any ofthe aforementioned solutions.

The invention further features kits for preparing or storing a donorcell, tissue, or organ for transplantation into a recipient (includingkits for preconditioning steatotic cells, tissues, or organs), the kitincluding a solution for reducing intracellular lipid storage materialof the donor cell, tissue, or organ and instructions for using thesolution(s) provided in the kit. Optionally, the solution within the kitfurther contains an agent that increases the expression of at least oneheat shock protein in a cell, tissue, or organ, such as cobaltprotoporphyrin or geranylgeranylacetone.

The invention further provides a device for preparing a cell, tissue, ororgan having excessive fat content for transplantation into a recipientby inducing heat shock in the cell, tissue, or organ. Desirably, such adevice contains any of the solutions of the invention, such as asolution for reducing intracellular lipid storage material of a cell,tissue, or organ as described herein. According to this invention, theinduction of heat shock may occur in vivo or ex vivo. For example, thedevice of the invention may increase the temperature of the tissue ororgan in a localized area by the emission of ultrasound or microwaves,for example. Alternatively, the device of the invention may have a heatexchanger that allows the cell, tissue, or organ to be contacted with asolution (e.g., defatting solution, saline, or blood) that has beenheated and that in turn induces heat shock in the cells of the donormaterial. Preferably, the device contains a heat exchanger that heatsthe cell, tissue, or organ to both 37° C. and 42° C. Accordingly, thecell, tissue, or organ being prepared using this device would bedefatted and heat shocked, either simultaneously or sequentially. Suchan exemplary device is shown in FIG. 1B.

In another aspect, the invention features a cell, tissue, or organprepared according to any one of the aforementioned methods involvingthe reduction of intracellular lipid storage material, heat shockpreconditioning, or both, and therefore includes isolated defatted donorcells, tissues, or organs that may be used for transplantation into arecipient.

In a final aspect, the invention features a method of transplanting acell, tissue, or organ, the method including (a) providing any of theaforementioned defatted cells, tissues, or organs; and (b) transplantingsuch a cell, tissue, or organ into a recipient.

By “lipid storage material” is meant any of a Variety of cellularsubstances that are soluble in nonpolar organic solvents. Such materialincludes, without limitation, triglycerides, cholesterol, cholesterolesters, free fatty acids, and phospholipids.

By “reducing intracellular lipid storage material” is meant decreasingan amount of lipid storage material in a cell, tissue, or organ byinducing catabolic metabolism of the lipid storage material byincreasing lipid export, lipid oxidation, or both from the cell, tissue,or organ. Typically, the intracellular lipid storage material of a donorcell, tissue, or organ is measured relative to the intracellular lipidstorage content of a control cell, tissue, or organ. In preferredembodiments, the lipid storage material of a donor cell, tissue, ororgan is reduced by at least 20% (and preferably 30% or 40%) as comparedto the lipid storage material of a control cell, tissue, or organ. Inother preferred embodiments, the lipid storage material is reduced by atleast 50%, 60%, and more preferably reduced by 75%, 80%, 85%, or even90% of the level of a control; with at least a 95% reduction in lipidstorage material as compared to a control being most preferred. Thelevel of lipid storage material is measured using conventional methods,such as those described herein. A reduction in the intracellular lipidstorage material of a cell, tissue, or organ is referred to asdefatting.

By “induce heat shock” is meant to elicit in a cell, tissue, or organ aresponse characteristic of the cell's, tissue's, or organ's naturalresponse to elevated temperatures. Typically, induction of heat shockpromotes the ability of a cell, tissue, or organ of the invention towithstand ischemia-reperfusion induced damage. According to thisinvention, heat shock induces the expression of various proteinsincluding heat shock proteins, such as HSP72, HSP70, HO-1, and HSP90.The expression of heat shock proteins may be increased by at least 10%,20%, preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%,or even more than 100% relative to such expression in a cell, tissue, ororgan in which heat shock has not been induced. Typically, heat shockinduction also decreases the proliferation and activation of T cellswithin the tissue or organ and decreases the production of inflammatorycytokines (e.g., IL-12, Il-10, IFN γ, and TNF α). Preferably, T cellproliferation or activation, or alternatively, the production ofinflammatory cytokines is decreased by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100% or even more than 100% relative to suchproliferation and activation, or alternatively such production, in acell, tissue, or organ in which heat shock has not been induced.Typically, heat shock is induced by increasing the temperature of thecell, tissue, or organ to a temperature ranging between 37° C. to 50°C., preferably between 38° C. and 45° C., more preferably between 40° C.and 43° C., and even more preferably between 42° C. and 43° C. Thetemperature of the cell, tissue, or organ may be increased using anymethod known in the art. Such temperature may be increased, for example,by contacting the cell, tissue, or organ with a solution that has beenheated, or alternatively, using ultrasound or microwaves. Optionally,the cell, tissue, or organ may be provided with the heat shock proteinor proteins by any method known in the art, including proteinmicroinjection or transfection.

By “ischemia-reperfusion related injury” is meant any damage, includingloss of viability, caused to a donor cell, tissue, or organ subsequentto a decrease in the availability of oxygen followed by a suddenincrease in oxygen levels. Ischemic or hypoxic conditions for thepurposes of the present invention are typically caused by (1) surgicalprocedures, which require temporary blood flow arrest, including forexample liver resection and vascular reconstruction, and (2) storage ofthe cell, tissue, or organ in the absence of a continuous supply ofoxygen. Such conditions allow for the generation of inflammatorymediators, reactive oxygen species, and nitric oxide, as well as theinfiltration of neutrophils, which can severely damage cells, tissues,and organs. The length of time necessary for ischemia-related damage istissue-dependent, and certain cells, tissues, or organs may be moresusceptible to hypoxic donations as a result of their high-energydemands.

By “cold-preservation related injury” is meant any damage caused to thecell, tissue, or organ caused by the storage of a cell, tissue, or organin hypothermic conditions for transplantation purposes. As an example,under hypothermic conditions, phospholipids forming the lipid bilayer ofthe cellular membranes undergo a phase change leading to a reduction influidity. As a result of this phase change, the cell fails to utilizeoxygen as efficiently, in a situation analogous to anoxic conditions.

By “anti-oxidant” is meant any agent that scavenges reactive oxygenspecies, which are generated in instances in which oxygen tension isincreased. Changes in oxygen tension may result from a transition fromanoxic to normoxic conditions, or from normoxic to supraphysiologicaloxygen tension. Examples of anti-oxidants include but are not limited toN-acetyl-cysteine, glutathione, allopurinol, S-adenosyl-L-methionine (aprecursor of glutathione), polyphenols (e.g., in green tea), free ironscavengers (e.g., deferoxamine), adenosine, or inhibitors of induciblenitric oxide synthase (iNOS) (e.g., N(G)-nitro-L-arginine methyl esterand aminoguanidine), cyclodextrin, superoxide dismutase (SOD), catalase,chlorpromazine, and prostacyclin.

By “reconditioning a cell, tissue, or organ for transplantation” ismeant restoring a cell, tissue, or organ, which is deemed unacceptablefor transplantation, into a transplantable form.

Although the most widely tested method of organ preconditioning isischemic preconditioning (induced by clamping major feeding vessels ofan organ), such methods may have at best a negligible effect on thesurvival of transplanted steatotic livers, which are more likely tomanifest ischemic injury in comparison with normal lean livers. Incontrast to the prior art, the present invention is particularly usefulfor the preconditioning of steatotic cells, tissues, and organs and istherefore advantageous for several reasons: (1) it will increase thedonor pool size, as severely steatotic organs (e.g., livers) are usuallydiscarded; (2) it will improve the outcome of patients who receive organtransplants with mild to moderate steatosis; (3) it will provide asimilar approach for a variety of organ systems prone to steatosisduring obesity, such as pancreatic β cells and cardiomyocytes; (4) itwill provide methods for preventing or limiting hepatic fibrosis, ashepatic steatosis often precedes fibrosis in degenerative liverdiseases; and (5) it will further optimize organ preservation techniquesand exploit the potential of long-term warm perfusion preservationtechniques. Furthermore, the metabolic preconditioning regimens of theinvention that reduce the lipid load and modulate the redox state ofcells (e.g., liver cells) will reduce the impact of I/R and prolong thepreservation time of donor livers.

Other features and advantages of the invention will be apparent from thefollowing Detailed Description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic diagram of a perfusion apparatus used to defatlivers. The liver is immersed in the perfusate solution and perfused viathe portal vein at a rate of 4 mL/min/g liver. The perfusate is heatedto 37° C. through a heat exchanger and oxygenated by passing through athin silicone tubular membrane exposed to 95% oxygen and 5% carbondioxide. A bubble trap is placed immediately before the perfusate entersthe liver.

FIG. 1B shows a schematic diagram of a perfusion apparatus used toinduce heat shock in a liver. The perfusate solution is heated to 42° C.through a heat exchanger and used to perfuse the liver via the portalvein.

FIGS. 2A-2D show the morphological appearance of cultured hepatocytesafter 7 days of plasma exposure. FIG. 2E shows a graph of theintracellular triglyceride levels in hepatocytes for conditions shown inFIGS. 2A-2D. Statistical differences were determined using ANOVA withTukey's post hoc test (n=11).

FIG. 3 shows the effect of defatting medium on cultured hepatocyteappearance after 2 days of defatting.

FIGS. 4A and 4B show the release of lactate dehydrogenase by culturedhepatocytes after I/R at 37° C. FIG. 4A shows the effect of hypoxic timebefore reoxygenation in steatotic and normal “lean” hepatocytes. FIG. 4Bshows the effect of defatting time on the response of steatotichepatocytes to I/R.

FIGS. 5A and 5B show the release of lactate dehydrogenase (LDH) bycultured hepatocytes in response to 12 hours of storage at 4° C.followed by rewarming at 37° C. FIG. 5A shows LDH activity after 12hours in University of Wisconsin (UW) solution at 4° C. and 12 morehours at 37° C. in medium. FIG. 5B shows the effect of defatting time onthe response of steatotic hepatocytes to cold storage followed byrewarming.

FIG. 6 shows the proportion of cytochrome c detected in the cytosolicfraction of hepatocytes after 12 hours of storage at 4° C. followed byrewarming at 37° C. Cytosolic cytochrome c is normalized to total(cytosolic+mitochondrial) cytochrome c.

FIGS. 7A and 7B show the effect of hepatocyte island size and steatosison hepatocyte viability after I/R. FIG. 7A shows intensity of calceinfluorescence per surface area over hepatocyte islands at various timepoints during I/R of steatotic hepatocytes co-cultured withnonparenchymal cells. FIG. 7B shows calcein fluorescence per surfacearea over hepatocyte islands at the 4 hour time point (1 hour of no flowfollowed by 3 hours of flow).

FIGS. 8A and 8B show that rats fed a choline and methionine-deficientdiet (CMDD) developed fatty livers. FIG. 9A shows the kinetics ofhepatic triglyceride (TG) accumulation in rats fed a CMDD for up to 6weeks. FIG. 9B shows the restoration of the hepatic TG content to normallevels upon return of CMDD animals to a regular diet.

FIG. 9 shows that defatting makes fatty donor livers suitable fortransplantation. Survival curves for rats receiving donor livers areshown. Livers were stored for 6 hours in UW solution prior totransplantation. CMDD refers to fatty liver recipients. CMDD+RF 3d or 7drefers to recipients receiving donor livers from CMDD fed rats followedby refeeding (RF) with a normal diet for 3 or 7 days, respectively.

FIGS. 10A and 10B show the effect of amino acids in the perfusate onliver triglyceride content after 3 hours of warn perfusion (panel A) andthe effect of perfusion time on liver triglyceride content using aminoacid-containing perfusate (panel B). Fatty livers from CMDD fed rats for6 weeks were perfused at 37° C. After 3 hours of perfusion, theremaining TG content is in the normal range (˜10 mg/g liver).

FIG. 11A is a series of bar graphs showing the levels of HSP72 asmeasured by ELISA, in livers harvested before heat shock preconditioning(HPc) (pre) or between 3 and 72 hours after HPc. Data shown are forHPc-fatty livers (n=6), sham HPc-fatty livers (n=3), and HPc-normallivers (n=7). Bars represent mean±SD. * P<0.05 compared to “pre” levels.#P<0.05 compared to levels at three hours. $P<0.05 compared to levels at6 hours. &P<0.05 compared to levels at 12 hours. §P<0.05 compared tolevels at 24 hours.

FIG. 11B is a series of immunoblots showing the protein expression ofHSP72, HO-1, and HSP90 in fatty livers. Data shown are representative ofthree rats in the HPc and one rat in the sham HPc groups.

FIG. 11C is a series of bar graphs representing the quantification ofprotein bands shown in FIG. 12B.

FIGS. 12A-12D are a series of bar graphs showing the effect of heatpreconditioning (HPc) on the levels of hepatic enzymes and inflammatorycytokines induced by the transplantation of fatty livers. Donor fattylivers were harvested 24 hours after HPc (▴) or sham HPc (∘), preservedin cold UW solution for 10 hours, and then transplanted into syngeneicanimals. ALT (FIG. 12A) and AST (FIG. 12B) activities in the serum ofthe recipient, as well as serum TNF-α (FIG. 12C) and IL-10 (FIG. 12D)levels, are shown as measured by ELISA. Data shown represent the mean±SDfor 6 rats. *P<0.05 between groups. **P<0.01 between groups.

FIGS. 13A-13H are a series of photographs showing the effect of heatpreconditioning on fatty liver transplantation. HPc prevents hemorrhageand confluent hepatocellular necrosis in fatty livers. The transplantedlivers from HPc (FIGS. 13B, 13D, 13F, and 13H) or sham-HPc (FIGS. 13A,13C, 13E, and 13G) donors were harvested 3 hours (FIGS. 13A, 13B, 13C,and 13D) or 24 hours (FIGS. 13E, 13F, 13G, AND 13H) afterrevascularization, and stained by hematoxylin and eosin. Severecongestion, hemorrhagic change (arrows), and areas of confluenthepatocellular necrosis (arrow heads) were seen in the sham-treatedgroup, with significant reduction of these findings seen in the HPcgroup at all histologic features considered reduction in extent anddegree of hemorrhagic injury was the most striking hallmark of the HPcgroup. Original magnification FIGS. 13A, 13B, 13E, and 13F, 40×; FIGS.13C, 13D, 13G, and 13H, 120×.

FIG. 14 is a graph showing the effect of heat preconditioning (HPc) onthe survival of recipients after fatty liver transplantation.Transplantation of cold preserved (for 10 hours) fatty livers wasperformed 24 hours after HPc (▴; n=12) or sham HPc (∘; n=12). Also shownare data for transplantation of cold preserved (for 10 hours) normallivers (▪; n=7). Differences among groups: fatty liver HPc vs. fattyliver sham HPc (P<0.005); normal liver vs. fatty liver sham HPc(P<0.01); normal liver vs. fatty liver HPc (not significant).

FIG. 15 is a series of immunoblots showing the protein expression ofHSP72 and HO-1 in hepatocytes, CD4⁺ T cells, and CD8⁺ T cells insteatotic rat liver after HPc (n=3), sham HPc (n=3), and GdCl₃ treatment(n=3). Livers were harvested 24 hours after treatment and CD4⁺ and CD8⁺T cells were separated by flow cytometry. The expression level of HSP72and HO-1 was analyzed by Western blot. 5 μg of total protein was loadedin each lane and the data is representative of three separateexperiments.

FIG. 16 is a graph showing the effect of heat shock preconditioning orGdCl₃ on serum heptic enzyme levels (ALT) after fatty livertransplantation. Donor livers were harvested 24 hours after HPc (n=5),GdCl₃ injection (n=5), or sham HPc (n=5), preserved in cold UW solutionfor 12 hours and then transplanted. Sera were collected from recipientrat up to 24 hours after hepatic revascularization and measured forlevels of ALT activity. Data are representative of 3 separateexperiments and show mean±SD for 5 rats. *p<0.05 compared to Sham group.**p<0.01 compared to Sham group.

FIGS. 17A-17F are photographs showing the effect of heat shockpreconditioning or GdCl₃ on the morphology of transplanted fatty livers.The transplanted livers from sham-heat shock preconditioned (17A and17D), heat shock preconditioned (17B and 17E), or GdCl₃ pretreated (17Cand 17F) donor were obtained 3 and 24 hours after revascularization, andstained by hematoxylin and eosin staining. Original magnification; 100×.

FIGS. 18A-18C is a series of graphs showing the effect of heat shockpreconditioning or GdCl₃ on the level of serum cytokines after fattyliver transplantation. Donor livers were harvested 24 hours after HPc(n=6), GdCl₃ injection (n=6) or sham HPc (n=6), preserved in cold UWsolution for 12 hours and then transplanted. Sera were collected fromrecipient rat up to 24 hours after hepatic revascularization andmeasured for levels of IL-12p70, TNF-α, and IL-10. Data arerepresentative of three separate experiments and show the mean±SD for 5rats. *p<0.05 compared to Sham group.

FIG. 19 is a bar graph showing the effect of heat shock preconditioningor GdCl₃ on myeloperoxidase in the liver after fatty livertransplantation. Donor livers were harvested 24 hours after HPc (n=6),GdCl₃ injection (n=6) or sham HPc (n=6), preserved in cold UW solutionfor 12 hours, and then transplanted. Livers were harvested fromrecipient rat 3 hours and 24 hours after hepatic revascularization andmeasured for levels of myeloperoxidase in liver tissues. Data arerepresentative of 2 separate experiments and show the mean±SD for 5rats. *p<0.05 compared to Sham group.

FIG. 20 is a graph showing the effect of heat shock preconditioning(HPc) or GdCl₃ on the survival of recipient rats after livertransplantation. Donor livers were harvested 24 hours after HPc, GdCl₃injection, or sham HPc, preserved in cold UW solution for 12 hours, andthen transplanted. Survival rate of recipient rats was monitored for upto 1 week after transplantation. *: p<0.01 compared to Sham group. **:p<0.001 compared to Sham group.

FIG. 21A is a series of agarose gel photographs showing the level ofmRNA expression of IFN-γ in liver CD4⁺ T cells purified from liver ofrats 24 hours after transplantation. Donor livers were harvested 24hours after HPc, GdCl₃ injection or sham HPc, preserved in cold UWsolution for 12 hours, and then transplanted. 24 hours aftertransplantation, CD4⁺ T cells were purified from liver lymphocytespooled from 3 rats of each group, and mRNA was isolated for RT-PCR.

FIG. 21B is a graph showing the level of IFN-γ production by liver CD4⁺T cells (5×10⁵/well) purified from rats 24 hours after transplantation.Cells were incubated in anti-CD3 mAb-coated 96-well plates for 48 hoursat 37° C. after which the culture supernatants were collected. Cytokineactivity in the culture supernatant was determined for the presence ofIFN-γ by ELISA. Data are representative of 3 separate experiments andshow mean±SD for 5 rats. *p<0.05 compared to Sham group. **: p<0.001compared to Sham group.

FIG. 22A is a series of agarose gel photographs showing the level ofIFN-γ mRNA in CD4⁺ T cells isolated from transplanted fatty liversfollowing pretreatment with cyclosporin A (CyA) treatment. mRNAexpression of purified lymphocytes isolated from liver of rats wasdetermined 24 hours after transplantation. Donor livers were harvested24 hours after sham HPc, HPc, and GdCl₃ injection, as well as 6 hoursafter CyA treatment, preserved in cold UW solution for 12 hours and thentransplanted. 24 hours after transplantation, CD4⁺ T cells were purifiedfrom liver lymphocytes pooled from 3 rats of each group, and mRNA wasisolated for RT-PCR.

FIG. 22B is a bar graph showing the level of hepatic enzyme levels intransplanted fatty livers following CyA pretreatment. Donor livers wereharvested 24 hours after sham HPc (n=6) and HPc (n=6), and 6 hours afterCyA treatment (n=6), preserved in cold UW solution for 12 hours, andthen transplanted. Sera were collected from recipient rats up to 24hours after hepatic revascularization and measured for levels of ALTactivity. Data are representative of 3 separate experiments and showmean±SD for 5 rats. *p<0.05 compared to Sham group. **: p<0.001 comparedto Sham group.

DETAILED DESCRIPTION

In general, the present invention provides methods, solutions, anddevices for the metabolic preconditioning of a donor cell, tissue, ororgan for surgical purposes, including transplantation. These methodsinvolve reducing the intracellular lipid storage material of cells,tissues, or organs thereby increasing their ability to withstandischemia/reperfusion injuries (I/R), cold-preservation injuries, orboth. If desired, heat shock may also be induced in the cells, tissues,or organs of the present invention. Accordingly, the metabolic and heatshock preconditioning methods described herein improve the outcome ofvirtually any transplant surgical procedures and reduce the risk ofpostoperative organ dysfunction to a level similar to that observed innonsteatotic organs (e.g., livers).

Ischemia-Reperfusion (I/R) Injury

Ischemia-reperfusion (I/R) injury is inevitable in complex surgicalprocedures, such as liver transplantation and liver resection. In thisregard, hepatic steatosis is a major risk factor of primary malfunctionof graft livers because steatotic livers are especially susceptible tosuch injury.

Metabolic Preconditioning

Based on our identification of critical branch points of the hepaticmetabolic network affected by lipid loading, we hereby provide methodsand solutions useful for reducing lipid storage in donor cells, tissues,or organs. To this end, we have shown that two strategies may be used toreduce the lipid load, namely (1) hormonal modulation and (2) amino acidsupplementation. With the ultimate goal of using lipid-loweringtechniques in the clinic, noninvasive methods for monitoring such“delipidization” processes may also be employed in the methods of theinvention. Exemplary monitoring methods include nuclear magneticresonance (NMR) and positron emission tomography (PET) forquantitatively assessing lipid load and metabolism; furthermore, ajudicious choice of probes may also be used alone or simultaneously tomonitor the quality of perfusion and the energy status of cells,tissues, or organs.

Optimization of a Metabolic Network

Lipids are typically stored in the liver as triglycerides and areremoved by catabolic action. When this occurs, one molecule oftriglyceride is broken down into one molecule of glycerol and threemolecules of fatty acids, after which fatty acids undergo β-oxidation inthe mitochondria to generate reducing equivalents, CO₂, and ketonebodies. Triglycerides can also be removed from the liver by export inthe form of lipoproteins.

The methods of the invention involve maximizing the sum of fluxesrepresented by β-oxidation and triglyceride export. Furthermore, thepresent methods involve maintaining the intracellular triglyceridesynthesis flux to a minimum. These three fluxes are related to eachother as well as to the other metabolic fluxes via the stoichiometry ofthe hepatic metabolic network, which imposes mass balance constraints tothe set of possible fluxes.

Optimization of Fluxes

The predicted optimum fluxes are induced experimentally by a combinationof mass action effects, for example, by altering amino acid levels inthe perfusate or culture medium, and hormones which favor fatty acidoxidation and export of triglycerides, e.g., glucagon, epinephrine,growth hormone, hepatocyte growth factor, thyroid hormone, leptin,adiponectin, metformin, and various glucocorticoid hormones. Thesteatotic hepatocyte culture system described herein is used in thisoptimization effort, and the most effective regimen is then utilized inthe steatotic perfused liver system. Results from the first studies areanalyzed and re-fed into a linear optimization routine in order togenerate other predicted optimum perfusate compositions, which are thenutilized for treating a donor cell, tissue, or organ. Going throughseveral iterations with this process, the levels of all components ofthe perfusate may be optimized. Other optimization methods, such asthose using empirical simplex algorithms may be used as well.

During the experiments, culture medium/perfusate samples are obtained atregular intervals and the intrahepatic content of triglycerides andglycogen determined as well. Cultured hepatocyte defatting experimentsare performed for 24-48 hours and liver perfusions up to 3 hours, whichis sufficient to assess the effect of the defatting procedure. Controlhepatocytes or livers from littermates are not defatted and instead usedto provide the initial values of lipid/glycogen content. Throughoutthese studies, metabolic flux analyses are performed to characterize thelipid lowering mechanisms, and determine whether the cellular metabolicstate returns to that found in normal nonsteatotic livers as the lipidload disappears. To help in the optimization aspects of defattingperfused livers, noninvasive fat measurement methods based on protonchemical shift nuclear magnetic resonance (NMR) imaging and positronemission tomography (PET) using 1-[¹¹C]-3-R,S-methylheptadecanoic acidas a tracer are used to follow the process of delipidization in realtime.

Defatting Solutions

Organ preservation and perfusate solutions are known in the art ascomprising a base solution that consists of a buffered physiologicalsolution, such as a salt solution or a cell culture-like basal medium,to which is added a variety of defined supplements. In a preferredembodiment, the defatting solution of the present invention also employssuch a base solution containing amino acids, ions (e.g., sodium ion,potassium ion, phosphate ion, calcium ion, magnesium ion, andbicarbonate ion), physiologic salts, impermeants, serum proteins and/orfactors, and sugars (e.g., glucose). In addition to the components ofthe base solution, the defatting solution of the present inventioncontains a novel combination of supplements that can be grouped into atleast two component categories. It can be appreciated by those skilledin the art that the components in each category may be substituted witha functionally equivalent compound to achieve the same result. Thus, thefollowing listed species of components in each component category is forpurposes of illustration, and not limitation.

A first component category, hormones, comprises a combination ofcomponents in a physiologically effective amount, which provide a meansto reduce the lipid content in a cell, tissue, or organ by increasinglipid oxidation and lipid export from the cell, tissue, or organ. Toinsure that this catabolic activity in the cell, tissue, or organ ismaintained, conditions characteristic of starvation and thus amenable tolipid reduction are provided. These conditions may include highconcentrations of catabolic hormones (e.g., glucagon, epinephrine,growth hormone, hepatocyte growth factor, thyroid hormone, leptin,adiponectin, metformin, or glucocorticoid hormones including for examplehydrocortisone, corticosterone, cortisol and dexamethasone) and lowconcentrations of anabolic hormones (e.g., insulin). The result of usingsuch a combination of hormones simulate conditions of starvation in amammal and as such, the lipid content of a cell, tissue, or organ iseffectively reduced through the oxidation and the export of lipids. Thehormones comprise from about 1×10⁻⁶% to about 3×10⁻⁵% by volume (w/v) ofthe novel combination of supplements, which are added to the basesolution in forming the defatting solution of the present invention.

A second component category, amino acids, comprises a combination ofcomponents in a physiologically effective amount, which provide a meansto supply the building blocks required for the synthesis ofapolipoproteins, which are subsequently incorporated into largerlipoproteins. These lipoproteins export triglycerides and other lipids(e.g., cholesterol, cholesterol esters, and phopholipids) outside of thecell, tissue, or organ. Such amino acids added to the defatting solutionmay include any of the essential nutritional amino acid such as alanine,arginine, aspargine, aspartate, cysteine, glutamate, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine; and acombination thereof. The amino acids comprise from about 0.01% to about1% by volume of the novel combination of supplements, which are added tothe base solution in forming the defatting solution of the presentinvention.

It will be appreciated by those skilled in the art that components inany one or more of the two component categories can have additionalfunctions desirable for the process according to the present invention.For example, amino acids contained in the defatting solution includecysteine in amounts which, besides functioning as a building block forlipoproteins, also function as antioxidant-preferred free radicalscavengers which scavenge toxic free radicals during the flushing andperfusing steps of the process. These toxic free radicals are generatedin instances in which oxygen tension is increased (e.g., transition fromanoxic to normoxic conditions, or from normoxic to supraphysiologicaloxygen tension). Other antioxidants, including for exampleN-acetyl-cysteine, glutathione, allopurinol, S-adenosyl-L-methionine (aprecursor of glutathione), polyphenols (e.g., in green tea), free ironscavengers (e.g., deferoxamine), adenosine, or inhibitors of induciblenitric oxide synthase (iNOS) (e.g., N(G)-nitro-L-arginine methyl esterand aminoguanidine), cyclodextrin, superoxide dismutase (SOD), catalase,chlorpromazine, and prostacyclin may be included, or used asfunctionally equivalent compounds, in the defatting solution of thepresent invention. If present, such antioxidants comprise from about0.01% to about 5.00% by volume of the novel combination of supplements,which are added to, and dissolved in, the base solution in forming thedefatting solution of the present invention.

In another embodiment of the present invention, the defatting solutionmay further comprise cytoprotective agents, which can prevent apoptosisof cells resulting from the production of ceramide, a bi-product oflipid accumulation. Such cytoprotective agents can include, for example,membrane-permeable peptidic caspase inhibitors, cyclosporin A, and theinhibitor of ceramide production L-cycloserine. Other agents such asvitamins (e.g., choline chloride, folic acid, myo-inositol, niacinamide,pantothenic acid, pyridoxal HCl, riboflavin, thiamine HCL), ions (e.g.,sodium chloride, potassium sulfate, sodium phosphate (monobasic), sodiumbicarbonate, calcium chloride, and magnesium sulfate), carbohydrates(e.g., glucose), and pH indicators (e.g., phenol red) may also beincluded in the defatting solution. Optionally, the defatting solutionmay also contain agents, which can decrease lipid peroxidation,neutrophil infiltration, microcirculatory alterations, and the releaseof proinflammatory mediators such as TNF-α. The addition of such agentswould provide a means to minimize any damage caused byischemia-reperfusion injury. Agents which can provide oncotic pressuremay also be added to the defatting solution, including, but not limitedto, albumin, hydroxyethyl starch, or any high molecular weight polymer.

In another embodiment of the present invention, to avoid the use ofsupraphysiological oxygen tension and perfusion flow rate, the defattingsolution contains one or more oxygen transporting compounds (“oxygencarrying agents”) that function to provide molecular oxygen foroxidative metabolism to the ischemically damaged and injured organ. Suchoxygen carrying agents are known to those skilled in the art to include,but not be limited to, hemoglobin, stabilized hemoglobin derivatives(made from hemolyzed human or bovine erythrocytes such as pyridoxylatedhemoglobin), polyoxethylene conjugates (PHP), recombinant hemoglobinproducts, perfluorochemical (PFC) emulsions and/or perfluorochemicalmicrobubbles (collectively referred to as “perfluorochemical”). Suchoxygen carrying agents comprise from about 0% to about 50% by volume ofthe novel combination of supplements, which are added to, and dissolvedin, the base solution in forming the defatting solution of the presentinvention; or about 0% to about 20% of the total defatting solution(v/v).

In a process for preparing the defatting solution according to thepresent invention, to a base solution is added and dissolved therein anovel combination of supplements that can be grouped in at least twocomponent categories comprising hormones and amino acids. Although thecomposition of the defatting solution for use with the process accordingto the present invention can vary by component and component ranges aspreviously described, a preferred formulation is set forth below inTable 1 for purposes of illustration and not limitation.

The defatting solution thus prepared has an osmolarity >280 mOsm butpreferably less than 600 mOsm, and in a preferable range of about 300mOsm to about 350 mOsm. The pH of the resuscitation solution istypically adjusted to a pH within a pH range of about 6.5 to about 7.8,and preferably in a pH range of 7.3 to 7.45. The defatting solution mayalso be heated to a temperature of 25 to 40° C., but preferably, isheated to 34 to 39° C. The solution may also be exposed to 20 to 100% O₂and 0 to 10% CO₂, but preferably 95% O₂ and 5% CO₂.

In still another embodiment, the defatting solution may further includeantioxidants, oxygen carrying agents, ions, carbohydrates, vitamins,agents that can provide oncotic pressure and pH indicators as indicatedin Table 1. TABLE 1 Exemplary composition of a perfusate solution fordefatting livers. Component Concentration* Salts and CarbohydratesSodium chloride 116 Potassium sulfate 2.3 Sodium phosphate, monobasic1.0 Sodium bicarbonate 26 Calcium chloride 1.9 Magnesium sulfate 0.81Glucose 5.6 Amino Acids Alanine 0.48 Arginine 0.72 Asparagine 0.78Aspartate 0.063 Cysteine 0.26 Glutamate 0.33 Glutamine 2.00 Glycine 0.38Histidine 0.27 Isoleucine 0.40 Leucine 0.40 Lysine 0.50 Methionine 0.10Phenylalanine 0.19 Proline 0.42 Serine 0.63 Threonine 0.40 Tryptophan0.049 Tyrosine 0.29 Valine 0.39 Hormones Insulin 20 μU/mL Glucagon 100pg/mL Epinephrine 250 pg/mL Hydrocortisone 150 ng/mL Anti-oxidants andCytoprotective Agents N-acetyl-cysteine 2.0 Adenosine 5.0 Glutathione3.0 Allopurinol 1.0 Vitamins and Others Hydroxyethyl starch 60.0 g/mLCholine chloride 7.1 × 10⁻³ Folic acid 2.3 × 10⁻³ Myo-inositol 11 × 10⁻³Niacinamide 8.2 × 10⁻³ Pantothenic acid 4.2 × 10⁻³ Pyridoxal HCl 4.9 ×10⁻³ Riboflavin 0.27 × 10⁻³ Thiamine HCl 3.0 × 10⁻³ Phenol red 31 × 10⁻³*All values are in mM except otherwise indicated.Heat Shock Preconditioning

In addition to the metabolic conditioning methods described above, wehave also investigated the protective mechanism of heat shockpreconditioning (HPc) on recipient survival in fatty livertransplantation. For the purpose of our experiments, we compared theeffects of such treatment with gadolinium chloride pretreatment (GdCl₃),and Cyclosporine A pretreatment (CyA) on I/R injury in an experimentalcholine- and methionine-deficient diet induced rat fatty livertransplantation model.

Our results show that the induction of heat shock by exposing donor ratsto brief whole body hyperthermia (10 minutes at 42.5° C.) significantlyimproved the survival rate post-transplantation in normal rats relativeto donor rats that had not been treated (>80% survival after one weekvs. <10%). Evaluating the survival of recipients receiving fatty liversat different times following HPc, the protective effect of HPc was mostsignificant when donors were transplanted 3-48 hours after HPc, with themaximal effect seen 6-48 hours after HPc. Histological evaluation 3 and24 hours after transplantation revealed that HPc significantly reducedhepatic inflammation and hepatocellular necrosis without affecting thesteatotic appearance of hepatocytes. We further showed that heat shockpreconditioning was concomitant with an induction in heat shock proteins(HSP72, HSP90, and heme oxygenase-1 (HO-1)) in donor livers, withexpression levels peaking 12 to 48 hours after HPc.

Attenuation of Cellular Component in I/R Injury by HPc

Experimental I/R injury involves a cascade of events initiated byreactive oxygen intermediates and ultimately resulting in graft invasionby neutrophils and lymphocytes. To this end, membrane-derived compounds(e.g., platelet-activating factor), cytokines (e.g., tumor necrosisfactor and macrophage inflammatory protein-2), and adhesion molecules(e.g., the CD18 family, intracellular adhesion molecule-1, andselectins) are thought to depend on the activation of Kupffer cells.Collectively, these factors play a pivotal role in the recruitment andactivation of neutrophils.

I/R injury has recently been demonstrated to occur in a biphasicpattern: an initial acute phase characterized by hepatocellular damage(at 3-6 hours) and a subacute phase characterized by massive neutrophilinfiltration (at 18-24 hours), in which the activation of CD4⁺ T cellsplays a central role.

CD4⁺ T cells are subdivided into at least two subpopulations based ontheir functional pattern of secreted cytokines, Th1 and Th2. Th1 cells,which secrete IFN-γ, TNF-α and GM-CSF, may represent the best candidatesfor mediating inflammation. Among the various T cell-secreted cytokines,IFN-γ and TNF-α are known to be potent activator of Kupffer cells andmay likely promote local secretion of TNF-α and IL-1, which in turnfacilitates the interaction between endothelial cells and neutrophils byactivating neutrophils directly or by inducing changes in surfaceadhesion molecules on endothelial cells. Furthermore, Th1-secreted IFN-γand GM-CSF may also act directly on neutrophils and enhance theirability to damage liver tissue.

Serum alanine aminoatransferase (ALT), serum cytokines, liver histology,and liver CD4⁺ T cells were next analyzed. As described above, I/Rinjury in the liver has been demonstrated to occur in a biphasicpattern: an initial acute phase, characterized by hepatocellular damageat 3-6 hours and a subacute phase, characterized by massive neutrophilinfiltration at 18-24 hours. Similarly, the liver I/R injury in ourmodel following transplantation demonstrated a biphasic pattern, namelyan acute and a subacute phase. While HPc protected transplanted liveragainst I/R injury both in the acute (3 hours) and the subacute (24hours) phase, pretreatment with GdCl₃ (a potent inhibitor of Kupffercell function) only protected I/R injury in the acute phase. However,both HPc and GdCl₃ prevented the serum release of IL-12, TNF-α, andIL-10 produced by Kupffer cells. Kupffer cells are a major source ofreactive oxidants and proinflammatory cytokines that promote neutrophilrecruitment and adhesion, and eventually lead to organ injury. Thus, ourresults show that HPc could improve the overall recipient survival ratefollowing transplantation while treatment with GdCl₃ does not.

Our results also demonstrate a key role for CD4+ T cells in liver I/Rinjury. HPc suppressed the IFN-γ production in liver CD4⁺ T cells 24hours after transplantation, while GdCl₃ did not. CyA also suppressedthe IFN-γ production in liver CD4⁺ T cells and decreased serum ALTlevels, an event associated with liver injury. Thus, our results showedthat liver CD4⁺ T cells are involved in the cascade leading to therelease of cytokines and the development of liver injury. Thus, ourresults showed that HPc protects from liver I/R injury by modulating theactivation of both Kupffer cells and liver T cells in steatotic livertransplantation in rat. Because GdCl₃ pretreatment did not suppressactivation of or IFN-γ production by liver T cells 24 hours aftertransplantation and because GdCl₃ pretreatment did not suppress MPOlevel of transplanted liver tissue or recipient survival rate aftertransplantation, T cell involvement may lie proximal to the activationof Kupffer cells. Furthermore, T cells may be critical for theamplification of primary Kupffer cell cytokine responses within theinitial phases of injury. Overall, our results indicate that heat shockprecondititoning may have great potential for clinical applications bypreventing the I/R injury that is associated with steatotic livertransplantation.

Based on the above results, heat shock preconditioning may be used, inaddition to metabolic conditioning, to prepare the cells, tissues, andorgans of the invention. Desirably, the cells, tissues, and organs havean elevated fat content, and even more desirably, such cells, tissues,and organs are steatotic. HPc may be induced by increasing thetemperature of the cell, tissue, or organ of the invention by at least1° C. for at least one minute. Typically, the temperature is increasedfor a period ranging between one minute to one hour, preferably betweenone minute and thirty minutes, and more preferably between one minuteand fifteen minutes. The temperature of the cell, tissue, or organ maybe increased to a temperature ranging between 37° C. to 50° C.,preferably between 38° C. and 45° C., more preferably between 40° C. and43° C., and even more preferably between 42° C. and 43° C. Such anincrease in temperature may be accomplished by any method known in theart. For example, HPc may result from heating the whole body of thedonor, or alternatively, may result from heating of the cell, tissue, ororgan ex vivo. In this regard, a steatotic liver may be harvested fromthe donor, heated for a period of 1 minute, placed in cold storage, andthen transplanted into a recipient mammal. Furthermore, the cell,tissue, or organ may be heated by localized heating, using microwave orultrasound treatment for example. Alternatively, HPc may be mediated bywarming the blood percolating the localized area of the cell, tissue, ororgan of interest. HPc may also be induced by contacting the cell,tissue, or organ with a solution (e.g., defatting solution) that hasbeen heated. Alternatively, the cell, tissue, or organ is contacted withan agent that increases the expression of at least one heat shockprotein. Exemplary heat shock proteins include HSP72; HSP70, HSP90, andHO-1. Agents such as cobalt protoporphyrin and geranylgeranylacetone areuseful for this purpose. Alternatively, the cell, tissue, or organ ofthe invention may be provided with a therapeutically effective amount ofat least one heat shock protein. In this regard, the heat shock proteinmay be provided as a recombinant polypepeptide (e.g., by means ofmircroinjection) or using an expression vector containing a nucleic acidsequence encoding a heat shock protein (e.g., a plasmid or a viralvector, such as an adenovirus, retrovirus, lentivirus, poxvirus,adeno-associated virus, herpes simplex virus, or vaccinia virus) by anystandard method known in the art. Using any of the above methods, theexpression of the heat shock protein is increased by at least 10%, 20%,preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, preferably99%, more preferably 100%, or even more than 100% relative to anuntreated control as measured by any standard method known in the art.

HSPs may protect the cell, tissue, or organ from I/R injury by severalmechanisms, namely by providing anti-oxidant functions, by maintainingthe patency of hepatic microcirculation, by inhibiting apoptosis insinusoidal endothelial cells and hepatocytes, or by downregulatinginflammation (e.g., by decreasing the production of inflammatorycytokines and by suppressing NF-κB activation and subsequent TNF-αproduction by Kupffer cells following I/R injury). In this particularregard, our results clearly show that increases in TNF-α and IL-10,observed as early as 3 hours after transplantation in the untreatedgroup, were dramatically reduced by HPc treatment.

The observed reduction in early cellular damage (as shown by thereduction in the levels of ALT and AST) may further reduce theinflammatory stimulus. Accordingly, heat shock preconditioning as taughtherein preferably decreases T cell proliferation, T cell activation, orboth (e.g., in CD4+ T cells) by at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, preferably 99%, more preferably 100%, or even morethan 100% relative to an untreated control. CD4+ T cells produceinflammatory cytokines, activate Kuffpner cells, and recruitneutrophils. As a result of such a reduction, the production ofcytokines, such as IL-10, IL-12, IFN-γ, and TNF-α, is decreased.Overall, our data suggests that HPc decreases the cellular damage andthe inflammatory response that occurs early after transplantation. Ifdesired, the cell, tissue, or organ that has undergone HPcpreconditioning according to the invention may further be contacted witha composition containing GdCl₃ or an agent that inhibits theproliferation, activation, or both of T cells (e.g., cyclosporine A orFK506). Optionally, the cell, tissue, or organ of the invention may becontacted with anti-TNF-α antibodies; FR167653, an agent that suppressescytokine generation and decreases hepatic IR injury; inhibitors ofKupffer cell activation, adenosine, and antioxidants (e.g.,α-tocopherol, lazaroid, and superoxide dismutase).

Donor Transplant Material

A donor cell according to the invention may be obtained from virtuallyany source, autologous or heterologous, including kidney, heart, liver,lung, intestine, pancreas, bone marrow, and eye. Similarly, a donortissue or organ includes, without limitation, kidney, heart, liver,lung, intestine, pancreas, bone marrow, and eye.

Regimen/Apparatus/Timing

Cells, tissues, and organs can be defatted by simple incubation with anysolution described herein, for example, the solution disclosed inTable 1. Any cell, tissue or organ in which reduction of intracellularlipid material is desirable, including, for example, the liver, thekidney, the pancreas, the heart, the lung, the small bowel, the brain,the eye, or the skin may be contacted or perfused with the defattingsolutions disclosed herein.

If desired, cells, tissues, or organs are perfused with a defattingsolution using the perfusion apparatus shown in FIGS. 1A and 1B. As aspecific example, the liver can be immersed in the perfusate solution(preferably the defatting solution described herein) and perfused viathe portal vein at a rate of 4 mL/min/g of liver. Perfusion rate canrange between 1 mL/min/g to 5 mL/min/g, but preferably, perfusion shouldtake place between 3 mL/min/g to 4 mL/min/g. The perfusate solution isheated to 37° C. (or 42° C. if HPc is desired) through a heat exchangerand oxygenated by passing through a thin silicone tubular membraneexposed to 95% oxygen and 5% carbon dioxide. A bubble trap may be placedimmediately before the perfusate enters the liver.

Cells, tissues, and organs can be treated with the defatting solutionaccording to standard methods for a period of time sufficient to enabledefatting, including, 10 minutes, 30 minutes, 1 hour, 2 hours, or morethan 2 hours. In preferred embodiments, a donor cell, tissue, or organis treated with a defatting solution for two to three hours.

If desired, heat shock may also be induced in the cell, tissue, or organhaving excessive fat content and may be prepared for transplantationusing the device of the invention. According to this invention, thedevice may contain a heat exchanger that increases the temperature ofthe solution that contacts the tissue or organ. The cell, tissue, ororgan may therefore be contacted with a solution (such as blood, saline,and preferably the defatting solution described herein) that has beenheated to 42° C. using the device described above (see FIG. 1B).Alternatively, heat shock may be induced in a cell, tissue, or organusing a device that increases temperature in a localized area of thetissue or organ. The cell, tissue, or organ may or may not be in thedonor (in vivo or ex vivo, respectively), and the increase intemperature may result from microwaves or ultrasound emitted from thedevice.

Assessment of Fat Content in Donor Cells, Tissues, or Organs

Fat content of donor cells, tissues, or organs is determined accordingto standard methods in the art. For example, the cell, tissue, or organmay be examined histologically or biochemically (using a biochemicalassay kit) to assess triglyceride content. Alternatively, ¹³³Xenonhepatic retention may also be used as an accurate index for fatty liverquantification (Ahmad et al., J. Nucl. Med. 20: 397-401, 1979; Yeh etal., J. Nucl. Med. 30: 1708-1712, 1989). A less invasive method is basedon the fact that the peak resonance frequency of ¹H nuclei of waterdiffers significantly from that of aliphatic carbons (—CH₂—); protonchemical shift magnetic resonance imaging proved to be a sensitive andaccurate way to evaluate the localization and quantity of fat depositsin liver and even bone marrow (Rosen et al., Radiology 169: 469-472,1985; Rosen et al., Radiology 169: 799-804, 1988).

Storage/Preservation

The defatting solutions of the invention can be used to store, preserve,and/or protect cells, tissues, or organs when these materials arebrought into contact with the solution. A specific embodiment of theinvention is for the preservation or storage of a human liver, or humanliver tissue or cells. Another embodiment of the invention is for thepreservation of a human heart or human heart tissue or cells. Theinvention contemplates the use of the defatting solutions to preservemammalian cells, tissues, organs, or portion thereof. If desired, heatshock may also be induced in the cells, tissues, or organs prior to, orduring, storage and preservation. In addition, the solutions can be usedto facilitate transplantation of organs, e.g., by perfusion of the organor tissue during the transplantation procedure. Preferably, the organ orportion thereof is maintained in the appropriate solution at all times.

The defatting solutions of the invention can be used to maintainviability of cells, tissues, or organs during storage, transplantation,or other surgery. The invention includes a method of storing cells,tissues, or organs comprising contacting a donor cell, tissue, or organ,with the solution of the invention, such that the in vivo and/or invitro viability is prolonged. The solutions permit maintenance ofviability of a cell, tissue, or organ (e.g., a liver, heart, or lung)for up to 24 hours. Use of the solutions of the invention results inimproved viability.

Kits

The present invention advantageously provides convenient kits for use bypractitioners in the art for conveniently preparing a donor cell,tissue, or organ for transplantation into a recipient. In a preferredembodiment, a kit of the invention will provide sterile componentssuitable for easy use in the surgical environment. A kit of theinvention may provide sterile, defatting solution for preparing a donorcell, tissue, or organ for transplantation into a recipient. Generally,such a kit will include a defatting solution or a HPc-inducing solutionas described herein in appropriate containers, and optimally packagedwith directions for use of the kit. For example, a kit of the inventioncan provide in an appropriate container or containers: (a) apredetermined amount of at least one defatting solution; (b) ifnecessary, other reagents; and (c) directions for use of the kit forcell, tissue, or organ treatment or storage.

Transplantation

Once a cell, tissue, or organ is processed using the proceduresdescribed herein, such donor material is transplanted into a recipient(e.g., a human) according to standard methods known in the art.Following metabolic preconditioning, HPc, or both, the cell, tissue, ororgan may be placed in cold storage and transplanted into the recipientmammal 3 to 48 hours after HPc and preferably between 6 to 48 hoursafter HPc.

The following experimental examples are provided for the purpose ofillustrating the invention, and should not be construed as limiting.

Modulation of Lipid Accumulation in Hepatocytes Cultured in Plasma

It has previously been shown that collagen-sandwiched adult rathepatocytes which are seeded and maintained in standard hepatocyteculture medium and then exposed to either rat or human plasma becomeseverely steatotic within 24 hours with a concomitant reduction inliver-specific functions (Matthew et al., Biotechnol. Bioeng. 51:100-111, 1995; Stefanovich et al., J. Surg. Res. 66: 57-63, 1996). Morerecently, we found that intracellular accumulation of lipids occursduring exposure to plasma if hepatocytes are cultured in a mediumcontaining high levels of insulin (e.g., similar to that found instandard hepatocyte culture medium or 500 mU/mL) prior to plasmaexposure (Chan et al., Biotechnol. Bioeng. 78: 753-760, 2002). On theother hand, hepatocytes cultured in medium containing low insulin levels(50 μU/mL) exhibited little triglyceride accumulation during subsequentplasma exposure. In addition, triglyceride accumulation could be furtherreduced by direct plasma supplementation with an amino acid cocktail asdisclosed in Table 1 (FIG. 2).

We also measured the expression of various liver-specific functions byhepatocytes exposed to plasma. We found that despite the tremendousaccumulation of intracellular lipids, amino acid supplementation to theplasma allows hepatocytes to maintain the production of albumin andurea, as well as cytochrome P450 activities to levels similar to or evenhigher than hepatocytes in standard culture medium (Washizu et al., J.Surg. Res. 93: 237-246, 2000; Washizu et al., Tissue Eng. 6: 497-504,2000; Washizu et al., Tissue Eng. 7:691-703, 2001). Thus, we concludedthat by culturing hepatocytes in high insulin-containing hepatocyteculture medium followed by exposure to plasma supplemented with aminoacids, we could obtain steatotic hepatocytes expressing high levels ofliver-specific function. Recalling that steatotic livers do notgenerally show impaired functions in the absence of stressfulconditions, these hepatocytes would appear to be a suitable model ofsteatotic liver.

In the next set of experiments, we induced steatosis in hepatocytes byexposing them to high insulin levels followed by plasma for 2 days, andattempted to defat them using the following conditions: plasmasupplemented with amino acids and low insulin levels; culture mediumcontaining high insulin (500 mU/mL); culture medium containing lowinsulin (50 μU/mL) levels. We then measured the fraction of remainingtriglycerides after 1 and 2 days of treatment. Low insulin-containingmedium almost completely removed intracellular triglycerides (FIG. 3 andTable 2). The triglyceride removal kinetic data in Table 2 was used tocalculate a defatting rate for each defatting condition. For thispurpose, the fraction of initial triglyceride remaining was plotted as afunction of time on a semi-log plot, which yielded linear curves (notshown). The slopes of these lines, which correspond to the first orderrate of decay or triglyceride clearance from the cells during defatting,are shown in Table 2 for each defatting condition tested. From thesevalues, we can predict the fraction of intracellular lipid remainingafter any treatment time using the simple equation:Triglyceride Fraction Remaining=100e^(−[RateConstant][TreatmentTime])  (equation 1) TABLE 2 1st InitialOrder Metabolic Rates for 1^(st) Day of Defatting Triglyceride Decay(μg/10⁶ cells/day) “Defatting” % Remaining* Rate Cst TriglycerideTriglyceride Ketone Body Medium Day 1 Day 2 (h⁻¹) Removal SecretionSecretion Plasma, 50 μU/mL 85 62 0.010 108 −32 130 insulin + amino acidsMedium, 500 74 52 0.014 170 194 47 mU/mL insulin Medium, 50 30 4 0.067273 384 96 μU/mL insulin*Initial intracellular triglyceride content was 583 ± 120 μg/10⁶ cells.

Using the low insulin-containing medium, which was the most efficient atdefatting, we can estimate that a treatment time of about 10 hours wouldbe sufficient to remove 50% of the intracellular triglycerides, and 28hours to remove 85%, the latter of which would correspond to normalizingthe triglyceride content of a severely steatotic liver. This was asurprising result considering that a limited number of defattingconditions were tried, and it is expected that further optimization ofthis protocol will significantly reduce these defatting times. It isimportant to note that liver-specific functions, as determined by thealbumin and urea secretion rates, were not reduced during exposure tothis medium.

To determine the mechanism of defatting, we measured the triglycerideand ketone body secretion rates in the medium. We found that both ofthese rates were higher in the low insulin compared to the highinsulin-containing medium. In hepatocytes continuously exposed toplasma, there was no net secretion of triglycerides, which probablyexplains the slower defatting rate. It is interesting to note that thetotal mass of triglycerides released into the medium exceeded the rateof defatting, especially in the low insulin-containing medium,suggesting that a significant part of the triglycerides released arisesfrom de novo synthesis in hepatocytes. Thus, it is anticipated thataddition of drugs which inhibit triglyceride or free fatty acidsynthesis (e.g. see Loftus et al., Science 288: 2379-2381, 2000) couldsignificantly accelerate the rate of triglyceride clearance fromhepatocytes. We also investigated the effects of leptin and hepatocytegrowth factor on the defatting process. In low insulin-containingmedium, these agents did not further enhance lipid removal. Somefat-reducing effects were seen in high insulin containing media, albeitnot as dramatic as the reduction observed by lowering the insulinconcentration.

Response of Steatotic Hepatocytes to Ischemia/Reperfusion

To investigate whether I/R injury correlates with the level oftriglyceride loading in hepatocytes, we studied the response of normaland steatotic hepatocytes to I/R. Steatotic hepatocytes were generatedby exposure to plasma supplemented with 500 mU/mL insulin and aminoacids for 2 days. I/R was induced by switching the cells to anatmosphere containing 90% N₂ and 10% CO₂ for various lengths of time,after which the cells were returned to normoxic conditions. Culturesupernatants were harvested 12 hours after restoration of normoxia forthe determination of lactate dehydrogenase release, a measure of celllysis. Lactate dehydrogenase activity in the supernatant was normalizedto that of dead controls (hepatocytes subjected to rapid freeze-thaw).We found that steatotic hepatocytes are more sensitive to I/R than leanhepatocytes (FIG. 4A). To determine whether the lipid content at thetime of I/R is what determines the sensitivity of cells to I/R,hepatocytes were defatted for different lengths of time prior to I/R.Cell lysis after I/R decreased as a function of defatting time (FIG.4B).

In order to provide additional evidence that the lipid load indeeddetermines the resistance of cultured hepatocytes to I/R, weinvestigated the effect of cold storage followed by rewarming onhepatocyte lysis. Hepatocytes were made steatotic by culturing in plasmafor 2 days, after which they were incubated in the UW solution at 4° C.for 12 hours. The cells were then returned to standard hepatocyteculture medium at 37° C. for 12 hours, and the release of lactatedehydrogenase in the medium was determined. Consistent with priorobservations, lactate dehydrogenase release correlated with the amountof intracellular lipids in the hepatocytes (FIGS. 5A and 5B). As apreliminary assessment of the potential mechanisms of death in this cellculture model, we measured cytochrome c release from the mitochondrialto the cytosolic fraction of the cells, an indicator of apoptosis.Cytochrome c was quantified on Western blots of cytosolic andmitochondrial fractions of hepatocytes subjected to different defattingregimen leading to varying triglyceride content at the time of I/R. Wefound that cytochrome c release was significantly correlated (p<0.006)with triglyceride storage in hepatocytes (FIG. 6), consistent with thegreater extent of cell death shown in FIGS. 5A and 5B.

Effect of Liver Nonparenchymal Cells on the Hepatocyte Response to I/Rin Co-Cultures

Since various in vivo studies suggest that Kupffer cells may beactivated by I/R and exacerbate the injury (Lichtman and Lermasters,Sem. Liver Dis. 19: 171-187, 1999), we investigated the effect ofnonparenchymal cells on the response of steatotic hepatocytes to I/R inmicropatterned co-cultures. Hepatocytes were patterned as islands ofsizes ranging from 36 to 490 μm on tissue culture dishes using stenciltechnology described by Folch et al. (J. Biomed. Mater Res. 52: 346-353,2000; Ann. Rev. Biomed. Eng. 2: 227-256, 2000). The nonparenchymal cellfraction obtained from another liver cell isolation was then seeded ontop of the hepatocytes. Nonparenchymal cells only attach to the vacantspaces left in-between the hepatocyte islands. Thus, one can increasedirect hepatocyte-nonparenchymal cell interactions by reducing the sizeof hepatocyte islands, and vice-versa (Bhatia et al., J. Biomed. Mat.Res. 34: 189-199, 1997; Bhatia et al., Biotechnol. Prog. 14: 378-387,1998; Bhatia et al., J. Biomater. Sci. Polym. Ed. 9:1137-1160, 1998).The cultures were then exposed to plasma supplemented with high insulinlevels and amino acids for 2 days to cause steatosis. Five minutesbefore starting the I/R experiment, 1 μM calcein acetoxymethyl ester wasadded to the cells for 5 minutes. This compound is specifically retainedand converted to brightly fluorescent calcein within viable cells andreleased upon membrane rupture at the time of cell death.

A small flow device made by micro-molding of polydimethylsiloxane asdescribed elsewhere (Folch and Toner, Biotechnol. Prog. 14: 388-392,1998) was placed on top of the cells to create a mini cell perfusionbioreactor. The bioreactor was perfused with medium saturated with 90%air/10% CO₂ for 1 hour. The flow was then stopped for 1 hour. Because ofthe low aspect ratio of the flow channel above the cells (1 cm long, 1cm wide, and 100 μm high), hypoxia occurs inside the flow channel withina few minutes, which mimics the situation in the actual liver when bloodflow is stopped. Flow was then restored and cells visualized for anadditional 5 hours. The I/R experiment was set up on thetemperature-controlled stage of an inverted fluorescence microscopefitted with a digital video camera and image analysis software toquantify the fluorescence intensity distribution of at regular timesintervals. Since in these experiments we were primarily interested inhepatocyte viability, the intensity of calcein fluorescence per surfacearea over hepatocyte islands only was measured, averaged for each islandsize, and normalized to that measured initially. Hepatocyte viability,based on the fraction of initial calcein fluorescence intensity,decreased as a function of time after reoxygenation and was lower in thesmaller hepatocyte islands (FIG. 7A). In addition, hepatocyte viabilitydecreased as a function of hepatocyte island size in co-cultures and waslower in co-cultures than in pure hepatocyte cultures (FIG. 7B). Thesedata strongly support the hypothesis that nonparenchymal cells havedeleterious effects on hepatocyte viability after I/R. The data alsoshow that steatotic hepatocytes are more sensitive to I/R than leanhepatocytes, confirming our earlier data based on lactate dehydrogenaserelease in static cultures.

Non-Invasive Imaging of Hepatic Lipid Metabolism

Non-invasive quantitation of hepatic lipid content and metabolism ispotentially very useful to optimize and monitor the effect of defattingregimens. Prior studies have shown that proton chemical shift nuclearmagnetic resonance (NMR) imaging can provide a quantitative measurementof the liver fat content (Rosen et al., Radiology, 154: 469-472, 1985).In these experiments, rats were either alcohol-fed or received anintraperitoneal injection of ethionine, a protein synthesis inhibitor,to cause lipid accumulation. The NMR signal intensity was directlyproportional to the hepatic triglyceride content measured using abiochemical assay (FIGS. 8A and 8B).

This technique is noninvasive and does not require the animal or patientto undergo any particular preparatory procedures, except for therequirement of immobilization, as the imaging time takes about 45minutes. More recently, we applied the same technique to non-invasivelydetermine fat distribution in bone marrow of human patients (Rosen etal., Radiology, 169: 799-804, 1988). Later on, we found that thedistribution of fat determined by this technique is a useful surrogatemarker to monitor the severity of Gaucher disease and the efficacy oftreatments against acute leukemia (Gerard et al., Radiology, 183:39-46,1992; Johnson et al., Radiology 182:451-455, 1992). This technique may,if desired, be combined with other techniques to determine microvascularflow distribution and ATP levels in tissue during liver perfusions.

We have also developed methods to determine metabolic fluxes through thetricarboxylic acid and gluconeogenic pathways using ¹³C-NMR spectroscopyand gas chromatography-mass spectroscopy, which we used to investigatemetabolic changes in burned rats and patients (Vogt et al., Am. J.Physiol. 266:E1012-1022, 1994; Vogt et al., Am. J. Physiol. 272:C2049-2062, 1997; Yarmush et al., J. Burn Care Rehabil 20: 292-302,1999). As part of these studies, we recently improved the mathematicalformalism used to determine fluxes from ¹³C isotopic distributions byimplementing “atom mapping matrices,” which allow one to quicklyoptimize labeling strategies and adapt the quantitative model for dataanalysis (Zupke et al., Anal. Biochem. 247: 287-293, 1997). Thistechnology is useful in analyzing metabolic pathways of fatty acidoxidation and metabolism, and independently verify metabolic fluxesobtained with the stoichiometric mass balance model.

Because there are currently no real-time imaging techniques using NMR tostudy carbon metabolism, positron emission tomography (PET) is typicallyused to non-invasively monitor regional metabolism in burned patients.For example, previous studies in our laboratory have demonstrated thatPET and parallel arterial sampling after bolus injection ofL-[methyl-¹¹C]methionine and 1-[¹¹C]-3-R,S-methylheptadecanoic acid canprovide less invasive, regional assessments of the protein syntheticrate and fatty acid oxidation rate, respectively, than traditionalapproaches (Zaknun, J. Nucl. Med. 36: 2062-2068, 1995; Hsu et al., Proc.Natl. Acad. Sci. U.S.A. 93: 1841-1846, 1996). In sum, we haveestablished that PET can be used to study carbon metabolism in healthyhuman subjects and animals, and that it holds promise for future invivo, non-invasive studies of the influences of physiological factorsand pharmacological manipulations on regional metabolism (Fischman etal., Proc. Natl. Acad. Sci. USA. 27: 12793-12798, 1998).

Defatting of Rat Livers Restores Survival of Recipients

To induce hepatic steatosis in rats, rats (age/weight), prior tosurgery, were fed a choline- and methionine-deficient diet (CMDD) for 6weeks as described by Nakano et al. (Hepatology (26): 670-678, 1997).Rats fed a CMDD exhibited a time-dependent increase in livertriglyceride (TG) content from ˜10 to 250 mg TG/g liver after 5-6 weeks(FIG. 8A). This accumulation was reversible, as returning the animalback to a regular diet caused the hepatic TG content to return to normal(FIG. 8B).

CMDD fed rats were returned to a regular diet for 3 or 7 days beforeharvesting the livers for transplantation. The donor livers were removedand stored as follows. After laparotomy, the bile duct of the liver wascannulated with a short polyethylene tube. Veins emptying into theportal vein and the hepatic artery were subsequently ligated anddivided, and the portal vein was divided at the level of the inferiormesenteric vein. To prepare the portal vein cuff, a short polyethylenetube was slipped over the vein and the vein everted over the tube. Theinfrahepatic vena cava and suprehepatic vena cava, including part of thediaphragm, were then transected. The liver is flushed withhetastarch-free UW solution and stored in a reservoir containing thesame for 6 hours at 0° C.

The donors were stored in a hetastarch-free UW preservation solution for6 hours at 4° C., and then transplanted into a recipient rat as follows.The recipient animal was prepared by cannulating the bile duct, clampingthe portal vein, and tying shut the other vessels. The liver was removedand discarded. The donor liver is placed orthotopically, thesuprahepatic vena cava anastomosed, and the cuffed portal vein wasinserted into the recipient's portal vein. Blood is then allowed to flowinto the donor liver, and the infrahepatic vena cava is anastomosed. Thebile duct is reconnected and wrapped around the omentum. The abdominalincision is then closed. This protocol mimics the clinical situationwhich typically requires that the liver be preserved in the UW solutionfor several hours while it is being transported from the donor to therecipient site.

As is shown in FIG. 9, the control animals receiving untreated fattylivers did not survive after 4 days. In contrast, recipients of defattedlivers showed a complete recovery of survival rate, with nostatistically significant difference in survival when compared torecipients receiving control (nonfatty) livers.

Metabolic Preconditioning of Steatotic Perfused Livers to Reduce TheirLipid Content

Based on our cell culture data, we tested the effect of warm perfusionwith buffer containing no insulin and high glucagon (10 ng/mL) on thetriglyceride content of steatotic livers. Donor livers were prepared fortransplantation and then perfused at 37° C. as follows. Steatotic liverswere obtained by feeding rats a CMDD for 6-7 weeks. The buffer alsocontained 3% bovine serum albumin in order to prevent tissue swelling.Perfusions were carried out at a flow rate of 4 mL/min/g liver, 37° C.,and using 95% O₂/5% CO₂ for 1-3 hours in a recirculating mode. Theperfusate solution consists of Minimal Essential Medium supplementedwith hydroxyethyl starch (6% w/v), amino acids, glucagon,hydrocortisone, and anti-oxidants.

The triglyceride content of livers was measured after the perfusion andcompared to that of unperfused livers from rat littermates. Initially,we compared buffer vs. amino acid-containing medium, and found asignificantly increased rate of triglyceride clearance in the presenceof amino acids (FIG. 10A). Using amino acid-supplemented medium, weinvestigated the kinetics of clearance during the first 3 hours ofperfusion, and found a linear relationship (FIG. 10B). After 3 hours,warm perfusion reduced the triglyceride content of fatty livers by 85%.These data demonstrate that warm perfusion can be used to reduce thehepatic lipid storage of fatty livers.

In addition, as is shown in FIG. 10B, the TG content decreased as afunction of time and the defatting process was largely complete after 3hours. It is likely that there are two major mechanisms of action of thedefatting regimen. First, the catabolic hormones glucagon andhydrocortisone, which are in the perfusate, favor the oxidation oflipids, more specifically fatty acids. Second, the amino acids in theperfusate provide the building blocks required for the synthesis ofapolipoproteins, which are then incorporated into the largerlipoproteins. These lipoproteins export TG and other lipids (e.g.cholesterol) outside of the cell.

It is interesting to note that, based solely on typical measured oxygenuptake rates of perfused livers, one would predict a maximum possiblerate of lipid oxidation about one order of magnitude less than observedin FIGS. 8A and 8B, suggesting that other pathways of defatting (e.g.export of triglycerides in the form of lipoproteins) are probably veryimportant in this process. In addition, using the triglyceride clearanceequation (equation 1) fitted to cell culture data earlier would predicta decrease to 82% of the original lipid load after 3 hours of treatmentwith low insulin medium (as compared to the 85% measured), suggestingthat our steatotic hepatocyte culture model closely reflects thebehavior of fatty livers, and thus can be used to rapidly screen formore effective defatting regimens.

To summarize, fatty livers are very sensitive to ischemia-reperfusionand cold preservation-related injuries, which makes them unacceptablefor liver transplantation. We hypothesized that removal of the excessfat storage from fatty livers can restore their ability to undergo livertransplantation. We obtained fatty livers from rats fed a CMDD for 6 wk,stored them in cold hetastarch-free UW solution for 6 hours, andtransplanted them into normal recipient rats. While recipient rats had a90% rate of survival after transplantation of control normal leanlivers, they all died when receiving CMDD rat livers. If CMDD rats werereturned to a normal diet for 3 or 7 days prior to donating livers,effectively reducing the fat content of the livers by 33% and 85%,respectively, the recipients survived at rates similar to the controls.Furthermore, we found that it is possible to eliminate excess fatstorage from fatty livers by short-term perfusion of fatty livers exvivo. These results support the notion that liver perfusion could beused to recondition fatty livers and make them suitable fortransplantation.

Heat Shock Preconditioning of Steatotic Livers IncreasesIschemic-Reperfusion Injury

We next investigated the effect of heat shock preconditioning (HPc) onrecipient survival in the rat fatty liver transplantation model. Fattyliver donor rats were exposed to brief whole body hyperthermia (10 minat 42.5° C.) and allowed to recover.

Heat Shock Increases the Expression of Heat Shock Proteins

We first characterized the dynamics of induction of HSPs (see FIGS.11A-11C). We compared HSP72 levels in livers from CMDD-fed and normallean rats up to 72 hours after HPc. In steatotic livers, HSP72 levelsmeasured by enzyme-linked immunosorbent assay (ELISA) increased until 12hours after HPc and were highest between 12 and 24 hours after HPc (FIG.11A). Interestingly, this induction occurred faster than in normal leancontrols, in which HSP72 levels peaked at 48 hours. Using western blotanalysis, we then analyzed HSP72, heme oxygenase-1 (HO-1) and HSP90contents in livers from CMDD-fed rats up to 240 hours after HPc (FIGS.11B and 11C). We detected HSP72 and HO-1, both inducible HSPs, as earlyas 3 hours after HPc. HSP72 levels were highest 6-24 hours after HPc,consistent with our ELISA data, while HO-1 was highest 12-48 hours afterHPc. HSP90, a constitutive HSP, was detectable in controls and did notchange until 12 hours after HPc, after which it increased to stabilize24-48 hours after HPc, and decreased afterwards. Overall, we found thatHPc induced heat shock proteins (HSP72, HSP90, and heme oxygenase-1) indonor livers, with levels peaking 12 to 48 hours after HPc. Forsubsequent transplantation studies, we chose to harvest donor livers 24hours after HPc because all HSPs were highly expressed at that timepoint.

Effects of HPc on Liver Injury and Serum Cytokines afterTransplantation.

Prior to transplantation, we stored donor livers in hetastarch-freeUniversity of Wisconsin (UW) solution for 10 hours at 4° C. Followingtransplantation in recipients, we measured serum levels of alanineaminotransferase (ALT) and aspartate transaminase (AST) (both of whichreflect hepatocellular injury) as well as serum levels of tumor necrosisfactor alpha (TNF-α) and interleukin (IL-) 10 (used as indexes ofsystemic inflammation) (see FIGS. 12A-12D). AST and ALT levels peaked 3hours after transplantation of sham-treated livers and remained elevateduntil at least the 12 hour time point. Transplantation of HPc liversmoderated the initial increase in ALT and AST, although the values werenot significantly different from controls after the 12 hour time point.In controls receiving sham-treated livers, TNF-α and IL-10 levelsdramatically increased 3 hours after transplantation. In contrast, HPctreatment almost completely abrogated the elevation in cytokine levels,suggesting inhibition of the inflammatory response.

Effects of HPc on Histology after Transplantation

Histologic examination of transplanted steatotic livers demonstratedsevere congestion and confluent hemorrhagic change 3 and 24 hours aftertransplantation (see FIGS. 13A, 13C, 13E, and 13G). Fulminanthepatocellular necrosis was also apparent 24 hours after transplantation(FIGS. 13E and 13G). In contrast, HPc livers displayed greatly reducedhemorrhagic injury and necrosis arising in a sparse pattern (FIGS. 13B,13D, 13F, and 13H). It is noteworthy that, in all cases, hepaticsteatosis was evident and that there were no qualitative histologicdifferences between HPc and sham-treated livers in that respect. Liverhistology of the two groups prior to transplantation also showed nodifference in the severity of steatosis.

Donor livers were subsequently harvested 24 hours after HPc, placed incold storage for 10 hours, and transplanted into normal rats. At 3 hourspost-transplantation, HPc reduced serum liver enzymes in the recipients,and almost completely suppressed the release of TNF-α and IL-10.Histological evaluation 3 and 24 hours after transplantation show thatHPc significantly reduced hepatic inflammation and hepatocellularnecrosis without affecting the steatotic appearance of hepatocytes.

Heat Shock Preconditioning Increases Transplantation Survival

We monitored the effect of HPc of donor fatty livers on the survival ofrecipient rats for up to 1 week (FIG. 14). 11 out of 12 recipientsreceiving sham HPc livers died of primary graft dysfunction within 3days following transplantation. In contrast, HPc resulted in recipientsurvival exceeding 80% (10 out of 12). This survival rate was comparableto that seen with normal lean livers (86% or 6 out of 7) that weretransplanted using the same protocol in the absence of heat shocktreatment. Thus, HPc induced tolerance of fatty livers to cold I/Rinjury associated with transplantation.

Effects of Recovery Time and HSP Levels after HPc on Survival Rate ofRecipient Rats after Transplantation

We next determined the sensitivity of recipient survival rate to therecovery time period after HPc. In these experiments, we harvested donorlivers between 3 and 72 hours after HPc or sham preconditioning, andstored the livers for 10 hours at 4° C. prior to transplantation. Thedata indicate that the protective effects of HPc were present as earlyas 3 hours after HPc, and reached their maximal effect at 6 hours afterHPc (Table 3, which shows the effect of recovery time after HPc of donorlivers on the survival rate of recipients). TABLE 3 Statistical Recoverysignificance period Survival rate on day 7 vs. 24 hr (hr) HPc Sham vs.sham recovery 3 33.3% (3/9) 0.0% (0/6) P < 0.05 P < 0.05 6 77.7% (7/9)5.0% (0/6) P < 0.01 N.S. 24* 83.3% (10/12) 8.3% (1/12) P < 0.005 — 48 83.3% (5/6) 16.7% (1/6) P < 0.05 N.S. 72  30.0% (3/10) 10.0% (1/10) N.S.P < 0.01*time course shown in FIG. 14

There was no significant difference in the survival rates among the 6,24, and 48 hours groups. However, the protective effect of HPc haddisappeared 72 hours after HPc, therefore suggesting that the maximalprotective effects of HPc occurs in a window of time between 6 to 48hours after HPc.

One week after transplantation, non-heat shocked control transplantsexhibited a survival rate <10%, while heat shocked fatty liverrecipients survived >80% of the time. Evaluating the survival ofrecipients receiving fatty livers at different times following HPcrevealed that the protective effect of HPc was significant when donorswere transplanted 3-48 hours after HPc, with the maximal effect seen6-48 hours after HPc. Accordingly, HPc is a promising avenue to salvagerejected donor fatty livers and enhance the survival rate of fatty liverrecipients. This technique could significantly increase the annual donorpool supply.

Induction of HSPs after HPc and GdCl₃

In order to detect the expression of such HSPs in T cells, western blotanalysis was performed in CD4⁺ or CD8⁺ T cells and compared to that ofhepatocytes in Sham, HPc, and GdCl₃ groups at the time of harvesting(FIG. 15). In the HPc group, HSP72 and HO-1 were not only expressed inhepatocytes, but also in CD4⁺ and CD8⁺ T cells. On the other hand,neither HSP72 nor HO-1 was detected in any of these cells in both theSham or GdCl₃-treated groups.

Effects of HPc and GdCl₃ on Liver Injury after Transplantation

Liver injury after liver transplantation was determined by assessing thelevels of serum ALT (FIG. 16) and by histological analysis (FIGS.17A-17F). Serum ALT level of the Sham group that underwent livertransplantation after 12 hours cold preservation demonstrated a biphasicpattern of liver injury that peaked at 3 hours and 24 hours,representing early acute and subacute damage, respectively. Incomparison, the GdCl₃-treated group of rats demonstrated that the ALTlevels 3 hours after transplantation were significantly lower than thatof Sham group. In addition, the HPc-treated group exhibited ALT levelsthat were significantly lower than that of the Sham group at 3 hours and24 hours after transplantation. Consistent with serum ALT activities,histologic examination of transplanted Sham group livers demonstratedsevere congestion and confluent hemorrhagic change 3 and 24 hours aftertransplantation (FIGS. 17A and 17D). Fulminant hepatocellular necrosiswas also apparent 24 hours after transplantation (FIG. 17D). Incontrast, GdCl₃ livers displayed reduced hemorrhagic injury 3 hoursafter transplantation, compared to the Sham group livers (FIGS. 17C and17F). Moreover, HPc livers displayed greatly reduced hemorrhagic injuryand necrosis both of which arose in a sparse pattern (FIGS. 17B and17E). Liver histology of the three groups prior to transplantationshowed no difference in the severity of steatosis.

Effects of HPc and GdCl₃ on the Serum Cytokine Levels afterTransplantation

Heat shock preconditioning is known to suppress the production ofcytokines, such as TNF-α, and to reduce the accumulation of neutrophilafter I/R injury in the liver. We measured serum levels of IL-12p70,TNF-α, and IL-10, which were produced mainly by Kupffer cells (FIGS.18A-18C). IL-12, TNF-α, and IL-10 peaked 3 hours after transplantationof Sham group livers and TNF-α levels also demonstrated a biphasicpattern peaking at 3 hours and 24 hours. Transplantation of HPc andGdCl₃ livers moderated the initial increase in IL-12, TNF-α, and IL-10significantly. Moreover, TNF-α levels in the HPc group 24 hours aftertransplantation were significantly suppressed relative to that of thecontrol group. In contrast, such levels in the livers of theGdCl₃-treated group were not significantly different from that of livercontrols after the 24 hours time point. Serum IL-4 or IFN-γ was notdetected in any group at any stage after transplantation.

Effects of HPc and GdCl₃ on the Neutrophil Accumulation in the Liverafter Transplantation

Next, we determined the neutrophil accumulation to measure MPO contentof the liver tissues (FIG. 19). In the Sham group, MPO contents wereincreased 3 times and 18 times at 3 hours and 24 hours followingreperfusion, respectively, compared with levels prior totransplantation. Three hours after transplantation, MPO levels of theSham, GdCl₃-treated, and HPc groups were almost similar with nosignificant difference. On the other hand, there was a significantdifference in MPO levels in the Sham group and HPc group 24 hours afterthe transplantation although there is no difference between the Shamgroup and the GdCl₃-treated group.

Effects of HPc and GdCl₃ on Survival Rate of Recipient Rats afterTransplantation

Donor fatty livers were HPc or GdCl₃ treated and the effect of suchtreatment on the survival of recipient rats was monitored for up to 1week after transplantation. Survival curves of rats that underwent livertransplants are shown in FIG. 20. In transplantation cases with Shamgroup livers, 11 out of 12 recipients died of primary graft malfunction3 days following transplantation. Despite improvements in serum ALTlevels and structural amelioration (as shown by histological analysis) 3hours after transplantation, there was no significant difference betweenthe recipient survival rate of Sham and GdCl₃-treated groups. Incontrast, the recipient survival rate of HPc group livers wasdramatically improved.

Liver injury after liver transplantation with cold preservation iscaused mainly by I/R injury. We next studied if HPc had an effect on thelevels of monokines released from Kupffer cells and if such levelsreduced neutrophil accumulation in the liver, in turn suppressing liverinjury. Pretreatment with GdCl₃ suppressed liver injury and TNF-α,IL-10, and IL-12 release 3 hours after transplantation. However, GdCl₃did not suppress liver injury or neutrophil accumulation 24 hours aftertransplantation. Moreover, GdCl₃ did not improve the recipient survivalrate. These results indicate that while suppression of Kupffer cellsimproved liver injury in the acute phase (3 hours aftertransplantation), the same was not true for the subacute phase (24 hoursafter the transplantation). Furthermore, liver injury during thesubacute phase was more critical in graft survival rate.

Effects of HPc and GdCl₃ on Liver T Cells after Transplantation

Our findings that HPc, in contrast to GdCl₃ (an agent that suppressesKupffer cell activity) improved both acute and subacute phase liverinjury suggest that other cells such as T cells may play important rolesin liver injury and HPc protection in the liver. We next examined theeffect of HPc on the relative numbers of CD4⁺ T cells and CD8⁺ T cellsin the liver after transplantation by flow cytometry (Table 4, whichshows the effects of HPc and GdCl₃ on the relative number of T cellsafter transplantation). TABLE 4 3 hr 24 hr naive Sham HPc GdCl₃ Sham HPcGdCl₃ CD3⁺CD4⁺ 5.2 ± 1.4 7.5 ± 1.8 4.7 ± 2.5 8.4 ± 2.7 8.9 ± 2.7 4.8 ±1.3* 7.2 ± 2.2 CD3⁺CD8⁺ 3.1 ± 1.5 4.8 ± 1.2 3.2 ± 1.0 4.2 ± 1.2 5.9 ±1.5 3.2 ± 0.9* 5.6 ± 2.3Data are expressed as mean ± SD (×10⁵).*P < .05 versus the Sham group.

There was no difference in the numbers of CD3⁺ CD8⁺ cells in the liverbetween the Sham, GdCl₃, and HPc groups at any stage followingtransplantation. On the other hand, CD3⁺ CD4⁺ cells appeared to decreasein numbers in the livers of the HPc group compared to that of thecontrol group 24 hours after transplantation.

To determine the functional difference in liver T cells between theSham, GdCl₃, and HPc groups, we purified T cells bearing CD3⁺ CD4⁺ andCD3⁺ CD8⁺ from lymphocytes of liver transplanted 24 hours and examinedthe expression of mRNA specific for IFN-γ and IL-4 by means of cytokineRT-PCR. As shown in FIG. 21A, the expression level of IFN-γ mRNA inisolated CD3⁺ CD4⁺ cells from C and GdCl₃ groups was much higher thanthat from HPc group, whereas that expression level in isolated CD3⁺ CD8⁺cells remained low in all three groups. The expression of IL-4 mRNA wasnot detected in any group.

We next examined cytokine production by liver T cells from livers 24hours after transplantation in response to immobilized anti-CD3 mAb. Asshown in FIG. 21B, IFN-γ production by liver CD4⁺ T cells from thelivers of the HPc group was significantly decreased compared with thatby CD4⁺ T cells from the livers of the Sham and GdCl₃ groups.

Effects of CyA Pretreatment of Donor Rat on Liver Injury afterTransplantation

We found that the levels of CD4⁺ T cells were suppressed in HPc donorlivers after transplantation. To determine whether the suppression ofliver T cells was responsible for the protective effect of HPc on liverinjury after transplantation, rats were injected i.v. with CyA, a potentT cell-deactivating-agent, 6 hours before the harvesting of donorlivers. As shown in FIG. 22A, the administration of CyA diminished theexpression of IFN-γ mRNA as much as HPc 24 hours after transplantation.We assessed liver injury based on the serum levels of ALT 24 hours afterthe liver transplantation. As shown in FIG. 22B, serum ALT levels inCyA-treated liver was significantly lower than that in the Sham group,but significantly higher than that of the HPc group. Taken together,these results suggested that, suppression of liver T cells is partlyresponsible for the protection of HPc on liver injury aftertransplantation.

The above experiments were performed using the following methods andmaterials.

Methods

Induction of Hepatic Steatosis in Donor Rats.

Several fatty liver rat models are available for experimental purposes.In addition to genetically obese animals (which have steatotic livers),the accumulation of lipids in animal models may be induced, for example,from alcohol administration, lipotrope diets, and choline and methioninedeficient diets. Regarding the choline and methionine deficiency model,which is used herein, choline and methionine are essential precursorsfor the synthesis of very low density lipoproteins. The lack of cholineand methionine in the diet therefore blocks the export of triglyceridesfrom hepatocytes, resulting in fat accumulation in the liver. Within afew weeks of such a diet, rats develop a severe-grade hepatic steatosis,predominantly macrovesicular, without any evidence of inflammationand/or fibrosis. Triglycerides are the main component of the accumulatedfatty droplets with an increased molar percentage of palmitic and oleicacids. Because of the pathological and biochemical similarities of thismodel relative to fatty livers in humans, particularly in cases of richcarbohydrate diets, we have chosen the choline- and methionine-deficientmodel to study ischemia-reperfusion injury in steatosis liver. We alsoused a syngeneic rat model of liver transplantation, which includes a 6to 12 hour period of cold preservation in UW solution. This experimentalprotocol was designed based on a typical liver transplantation procedurein a clinical setting requiring that the donor liver be stored andtransported in ice-cold UW solution for several hours. An inbred strainof rats was used to eliminate the effects of allogeneic rejection. Insum, such an experimental model was the most suitable model to study theI/R injury in the steatotic liver transplantation.

All procedures with animals were approved by the Subcommittee onResearch Animal Care, Massachusetts General Hospital and in accordancewith National Research Council guidelines. Male Lewis rats (CharlesRiver, Wilmington, Mass.) weighing 280 to 320 g were housed in a 12hours day-light cycle and allowed free access to food and water. Toinduce fatty liver, the rats were CMDD-fed (Test Diet, Richmond, Ind.)for 40 to 44 days.

Experimental Groups and Treatments of Donor Rats

Donor animals were divided into three groups; heat shock preconditioning(HPc) group, sham HPc (Sham) group, and gadolinium chloride (GdCl₃)group. In the HPc group, rats were anesthetized and placed in awaterproof bag that was then immersed in a 43° C. water bath to elevatethe core body temperature (measured via a rectal digital thermometer) to42-42.5° C. Animals were maintained at that temperature for 10 min andthen removed from the warm bath. Animals then received 10 ml/kgintraperitoneal saline injection and were allowed to recover with freeaccess to food and water. Animals in the Sham and GdCl₃ groups underwentthe same procedures except that they were immersed in a 37° C. bath(sham HPc). In the GdCl₃ group, Kupffer cell deactivation was achievedby two 20 mg/kg GdCl₃ (Sigma, St. Louis, Mo.) intravenous injection 48and 24 hours before donor liver harvesting. In the Sham and HPc groups,sterile nonpyrogenic saline was used as instead of GdCl₃ solution. Donorlivers were harvested 24 hours after HPc or sham HPc. In someexperiments, rats were pretreated with 5 mg/kg i.m. Cyclosporine A (CyA;Sigma) 6 hours before donor liver harvesting.

Donor Liver Removal, Preservation, and Transplantation

Isogenic orthotopic liver transplantation was performed as described byKamada and Calne (Kamada et al., Transplantation 28:47-50, 1979). Donorlivers were harvested 3, 6, 24, 48, or 72 hours after HPc. Briefly, thebile duct was cannulated with a short intraluminal polyethylene stent.Veins emptying into the portal vein and the hepatic artery were ligatedand divided, and the portal vein divided at the level of the inferiormesenteric vein. The infrahepatic and suprahepatic vena cava, includingpart of the diaphragm, were transected. The liver was flushed with 10 mLcold saline containing 50 U heparin and 5 ml hetastarch-free Universityof Wisconsin solution (Sumimoto et al., Transplantation 48:1-5, 1989)and subsequently stored for 6-12 hours at 4° C. After cold storage,orthotopic liver transplantation was performed without hepatic arteryreconstruction. The donor liver was flushed with 6 ml cold Ringer'ssolution, the suprahepatic vena cava anastomosed with a 7-0 nylonrunning suture, and the portal vein anastomosed using the cufftechnique. Blood was allowed to flow into the donor liver, and theinfrahepatic vena cava anastomosed using the cuff technique. Afterrevascularization of the graft, the rat was given 8 ml/kg Ringer'ssolution and 2 mL/kg 7% w/v NaHCO₃ intravenously, and intramuscularinjections of 80 mg/kg penicillin and 100 mg/kg streptomycin. The bileduct was connected and wrapped around the omentum. Anhepatic time rangedfrom 14 to 16 minutes. For survival studies, the animals were returnedto standard housing facilities and monitored for up to one week. In thecase of animals used for biochemical and histological studies, animalswere sacrificed 3 hours, 6 hours, 12 hours, or 24 hours aftertransplantation. Blood samples were collected from the hepatic veindraining the left lateral lobe, as previously described (Kamada et al,supra).

Liver Triglyceride Content

Liver tissue was sonicated in 20 volumes of 0.25 M sucrose, 50 mMTris-HCl, 1 mM EDTA for 1 min at 4° C. Triglyceride concentration in thehomogenate was measured using a commercial kit (Sigma Chemical, St.Louis, Mo.).

Western Blot Analysis for HSP72, HSP90, and HO-1

Donor rats were sacrificed up to 240 hours after HPc. Liver tissue washomogenized in 4 volumes of 0.25 M sucrose for 30 seconds at 4° C. Liverproteins were separated by sodium dodecylsulfate-polyacrylamide gelelectrophoresis and transferred to polyvinylidene difluoride membranes(Sigma Chemical). Antibodies to detect HO-1 (1:2,000), HSP 72 (1:1,000),and HSP90 (1:1,000) were mouse monoclonals from Stressgen (Victoria,British Columbia, Canada). The secondary antibody was aperoxidase-conjugated goat-anti mouse IgG (Stressgen) diluted 1:10,000.Protein signals were visualized by chemiluminescence (Pierce, Rockford,Ill.) and recorded on a GS282 Scanner (BioRad, Hercules, Calif.).

ELISA for HSP72, TNF-α and IL-10

HSP72 levels in the liver homogenates and TNF-α and IL-10 levels inserum were determined by ELISA. HSP72 was analyzed using a commercialkit (Stressgen). TNF-α and IL-10 were analyzed using R&D Systems(Minneapolis, Minn.) monoclonal antibodies according to themanufacturer's instructions.

Biological Assays

To assess liver injury, alanine aminotransferase (ALT) was measured inserum samples using a commercially available kit (Sigma). TNF-α, IL-10,IL-4, IL-12, and IFN-γ levels in serum and cell culture supernatant weredetermined by Enzyme-linked immunosorbent assay (ELISA). ELISA forIL-12p70 was performed using Biosource (Camarillo, Calif.) kit. ELISAsfor others were performed using R&D systems (Minneapolis, Nebr.) mAbsaccording to the manufacture's instructions.

Blood Chemistry

Blood samples were collected from the hepatic vein draining the leftlateral lobe (Yoshioka et al., Hepatology 27:1349-1353, 1998). To assessthe extent of liver injury, ALT and AST were measured in serum using acommercial kit (Sigma Chemical).

Preparation of Liver Lymphocytes and Enrichment of CD4⁺ and CD8⁺ T Cells

Three hours or 24 hours after liver transplantation, transplanted liverwere perfused with sterile PBS through the portal vein to wash out allremaining peripheral blood and then meshed with stainless steel mesh.After the coarse pieces were removed by centrifugation at 50 g for 1min, the cell suspensions were again centrifuged, resuspended in 8 mL of45% Percoll (Sigma), and layered on 5 mL of 67.5% Percoll. The gradientswere centrifuged at 600 g at 20° C. for 20 min. Lymphocytes at theinterface were harvested and washed twice with PBS. CD4⁺ or CD8⁺ T cellswere purified by Rat T cell CD4 or CD8 column kit (R&D systems,Minneapolis, Nebr.) from the harvested liver lymphocytes. The purity ofsorted cells was more than 95%.

Flow Cytometry Analysis

For 3-color analysis, liver lymphocytes were incubated with saturatingamounts of phycoerythrin-conjugated anti-rat CD3α mAb (Pharmingen, SanDiego, Calif.) and fluorescein isothiocyanate-conjugated anti-rat CD4mAb (Pharmingen) for 30 min. Cells were analyzed with a FACSCalibur flowcytometer (Becton Dickinson, San Jose, Calif.). We carefully gated cellsby forward and side light scattering for the liver lymphocytes. The datawere analyzed using CyQuest software (Becton Dickinson).

Reverse Transcription-Polymerase Chane Reaction (RT-PCR)

Total RNA was extracted by the acid guanidium-phenol-chloroform methodfrom Isolated CD4⁺ and CD8⁺ T cells. Complementary DNA (cDNA) synthesisand polymerase chain reaction (PCR) were performed using a complementaryDNA cycle kit (Invitrogen Corp., San Diego, Calif.). The PCR wasperformed on a PCR thermal cycler (Applied Biosystems, Foster city, CA).PCR cycles were run for 30 sec at 94° C., 30 sec 54° C., and 30 sec at72° C. with 30 cycles. The specific primers were as follows: IL-4 sense,5′-GAA CCA GGT CAC AGA AAA AGG-3′ (SEQ ID NO: 1); IL-4 antisense, 5′-CTGCAA GTA TTT CCC TCG TAG G-3′ (SEQ ID NO: 2); IFN-γ sense, 5′-CAC GAA AATACT TGA GAG CC-3′ (SEQ ID NO: 3); IFN-γantisense, 5′-TCT CTA CCC CAG AATCAG CACC-3′ (SEQ ID NO: 4). The PCR product was subjected toelectrophoresis on a 1.5% agarose gel (Life Technologies).

Lymphocyte-Associated Cytokine Assays

Liver lymphocytes were obtained by the same method previously described.Tissue culture 96-well plates were incubated overnight at 4° C. with 50mg/mL anti-CD3ε mAb (Pharmingen). The plates were then washedthoroughly. The harvested lymphocytes (5×10⁵/well) were incubated in theanti-CD3ε mAb-coated plates for 48 hours. IFN-γ and IL-4 levels in theculture supernatants were determined by ELISA.

Assessment of Neutrophil Infiltration

The presence of myeloperoxidase (MPO), enzyme specific for neutrophil(and some macrophages), was used as an index of intrahepatic neutrophilaccumulation. Briefly, the frozen tissue was thawed weighed, and placedin 4 mL iced 0.5% hexadecyltrimethylammonium bromide and 50 mM potassiumphosphate buffer solution with the pH adjust to 5. Each sample was thenhomogenized for 30 sec and centrifuged at 12000 g for 20 min at 4° C.Supernatants were then mixed with hydrogen peroxide-sodium acetate andtetramethyl-benzidine solutions. The change in absorbance was measuredby spectrophotometry at 450 nm. One unit of MPO activity was defined asthe quantity of enzyme degrading 1 μM peroxide per minute at 25° C. pergram of tissue.

Histology

Animals were sacrificed before or 24 hours after HPc, and 3 or 24 hoursafter transplantation. Livers were fixed in 10% buffered formalin,embedded in paraffin, thin-sectioned, and stained with hematoxylin andeosin for transmission brightfield microscopic examination. Steatosiswas graded semiquantitatively as described elsewhere (Koneru et al.,Transplantation 73:325-330, 2002 and Adam et al., Transplant Proc.23:1538-1540, 1991).

Statistical Analysis

Data are expressed as means±SD. Difference among groups were determinedusing ANOVA and post hoc Tukey's test, except for survival studies,where the generalized Wilcoxon test was used. Differences were deemed tobe significant when P<0.05.

Other Embodiments

All publications mentioned in this specification are hereby incorporatedby reference to the same extent as if each independent publication orpatent application was specifically and individually indicated to beincorporated by reference.

1. A method for preparing a donor cell, tissue, or organ fortransplantation into a recipient, said method comprising reducingintracellular lipid storage material of said cell, tissue, or organ. 2.The method of claim 1, wherein a donor cell is prepared.
 3. The methodof claim 1, wherein a donor tissue is prepared.
 4. The method of claim1, wherein a donor organ is prepared.
 5. The method of claim 1, whereinsaid cell is a liver cell, said tissue is a liver tissue, or said organis a liver.
 6. The method of claim 1, wherein said method comprisescontacting said cell, tissue, or organ with a solution that increasesoxidation of a lipid; increases export of a lipid from said cell,tissue, or organ; or both.
 7. The method of claim 1, wherein saidintracellular lipid storage material is a triglyceride, a cholesterol, acholesterol ester, or a phospholipid.
 8. The method of claim 1, whereinsaid method results in reducing an ischemia-reperfusion injury in saidcell, tissue, or organ upon transplantation into a recipient.
 9. Themethod of claim 1, wherein said method results in reducing acold-preservation-related injury in said cell, tissue, or organ upontransplantation into a recipient.
 10. The method of claim 1, whereinsaid method reconditions a steatotic cell, tissue, or organ.
 11. Themethod of claim 10, wherein said steatotic cell is a liver cell, saidsteatotic tissue is liver tissue, or said steatotic organ is a liver.12. The method of claim 1, further comprising inducing heat shock ofsaid cell, tissue, or organ.
 13. The method of claim 12, wherein saidinducing is the result of increasing the temperature of said cell,tissue, or organ by at least 1° C. for at least one minute.
 14. Themethod of claim 13, wherein said temperature is increased for a periodranging between one minute and one hour.
 15. (canceled)
 16. (canceled)17. The method of claim 13, wherein said temperature of said cell,tissue, or organ is increased to a range between 37° C. and 50° C.18-20. (canceled)
 21. The method of claim 13, wherein said increasing ofsaid temperature is the result of heating the whole body of the donor ofsaid cell, tissue, or organ.
 22. The method of claim 13, wherein saidincreasing of said temperature is the result of heating a localized areaof the donor including said cell, tissue, or organ.
 23. The method ofclaim 13, wherein said heating is mediated by microwave or ultrasoundtreatment.
 24. The method of claim 22, wherein said heating is mediatedby warming the blood percolating said localized area.
 25. The method ofclaim 13, wherein said increasing of said temperature is the result ofheating said cell, tissue, or organ ex vivo.
 26. The method of claim 12,wherein said inducing is the result of contacting said cell, tissue, ororgan with an agent that increases the expression of at least one heatshock protein in said cell, tissue, or organ.
 27. The method of claim26, wherein said agent is cobalt protoporphyrin orgeranylgeranylacetone.
 28. The method of claim 26, wherein said cell,tissue, or organ is provided with at least one expression vectorcomprising a nucleic acid sequence encoding a heat shock protein. 29.The method of claim 1, further comprising administering a heat shockprotein to said cell, tissue, or organ.
 30. The method of claim 26,wherein said heat shock protein is selected from the group consisting ofHSP72, HSP70, HO-1, and HSP90. 31-35. (canceled)
 36. The method of claim1, further comprising contacting said cell, tissue, or organ with acomposition comprising gadolinium chloride (GdCl₃).
 37. The method ofclaim 1, further comprising contacting said cell, tissue, or organ witha composition comprising an agent that inhibits the proliferation,activation, or both of T cells.
 38. The method of claim 37, wherein saidagent is selected from the group consisting of cyclosporine A (CyA) andFK506. 39-42. (canceled)
 43. A solution for reducing intracellular lipidstorage material of a donor cell, tissue, or organ comprising acatabolic hormone and an amino acid, wherein said catabolic hormone isselected from the group consisting of glucagon, epinephrine, growthhormone, hepatocyte growth factor, leptin, adiponectin, metformin,thyroid hormone, and a glucocorticoid hormone and wherein said aminoacid is selected from the group consisting of alanine and glutamine.44-62. (canceled)
 63. A method for preparing a donor cell, tissue, ororgan for transplantation into a recipient, said method comprisingcontacting said donor cell, tissue, or organ with the solution of claim43. 64-87. (canceled)